Antibodies to oncostatin M receptor

ABSTRACT

The invention provides characterization of the disease and cancer-associated antigen, OSM-R.beta. The invention also provides modulators of OSM-R.beta, including a family of monoclonal antibodies that bind to antigen OSM-R.beta, and methods of diagnosing and treating various human cancers and diseases associated with OSM-R.beta.

TECHNICAL FIELD

This invention is in the fields of biology and immunotherapy. Morespecifically, it concerns a receptor for Oncostatin M (“OSM”), and itsmodulators such as polyclonal and monoclonal antibodies and otherpolypeptides or small molecules that bind to the oncostatin M receptorbeta subunit (OSM-R.beta). The invention further provides for thediagnosis and/or treatment of a variety of human diseases and cancersassociated with OSM using agonists, antagonists, modulators and peptidesthat bind to OSM-R.beta, including anti-OSM-R.beta antibodies. Thepresent invention also relates to the use of a modulator of OSM-R.betasuch as an anti OSM-R.beta antibody in the manufacture of a medicamentfor the treatment or prophylaxis of an inflammatory arthropathy orinflammatory disorder, or other human diseases and cancers associatedwith OSM-R.beta, and the use of OSM-R.beta antibodies in screening forsuch modulators.

BACKGROUND OF THE INVENTION

In addition to their known uses in diagnostics, antibodies have beenshown to be useful as therapeutic agents. For example, immunotherapy, orthe use of antibodies for therapeutic purposes has been used in recentyears to treat cancer. Passive immunotherapy involves the use ofmonoclonal antibodies in cancer treatments. See for example, Cancer:Principles and Practice of Oncology, 6^(th) Edition (2001) Chapt. 20 pp.495-508. These antibodies can have inherent therapeutic biologicalactivity both by direct inhibition of tumor cell growth or survival andby their ability to recruit the natural cell killing activity of thebody's immune system. These agents can be administered alone or inconjunction with radiation or chemotherapeutic agents. Rituximab andTrastuzumab, approved for treatment of non-Hodgkin's lymphoma and breastcancer, respectively, are two examples of such therapeutics.Alternatively, antibodies can be used to make antibody conjugates wherethe antibody is linked to a toxic agent and directs that agent to thetumor by specifically binding to the tumor. Gemtuzumab ozogamicin is anexample of an approved antibody conjugate used for the treatment ofleukemia. Monoclonal antibodies that bind to cancer cells and havepotential uses for diagnosis and therapy have been disclosed inpublications. See, for example, the following patent applications whichdisclose, inter alia, some molecular weights of target proteins: U.S.Pat. No. 6,054,561 (200 kD c-erbB-2 (Her2), and other unknown antigens40-200 KD in size) and U.S. Pat. No. 5,656,444 (50 kD and 55 kDoncofetal protein). Example of antibodies in clinical trials and/orapproved for treatment of solid tumors include: Trastuzumab (antigen:180 kD, HER2/neu), Edrecolomab (antigen: 40-50 kD, Ep-CAM), Anti-humanmilk fat globules (HMFG1) (antigen>200 kD, HMW Mucin), Cetuximab(antigens: 150 kD and 170 kD, EGF receptor), Alemtuzumab (antigen: 21-28kD, CD52), and Rituximab (antigen: 35 kD, CD20).

The antigen targets of trastuzumab (Her-2 receptor), which is used totreat breast cancer, and cetuximab (EGF receptor), which is in clinicaltrials for the treatment of several cancers, are present at somedetectable level on a large number of normal human adult tissuesincluding skin, colon, lung, ovary, liver, and pancreas. The margin ofsafety in using these therapeutics is possibly provided by thedifference in the level of expression or in access of or activity of theantibody at these sites.

In addition to cancer targets, antibody therapeutics have also beenshown to be effective against chronic inflammation and other immunedisorders. An example of an antibody therapeutic approved for treatmentof immune disorders is Infliximab (antigen: TNFα).

Another type of immunotherapy is active immunotherapy, or vaccination,with an antigen present on a specific cancer(s) or a DNA construct thatdirects the expression of the antigen, which then evokes the immuneresponse in the individual, i.e., to induce the individual to activelyproduce antibodies against their own cancer. Active immunization has notbeen used as often as passive immunotherapy or immunotoxins.

Several models of disease (including cancer) progression have beensuggested. Theories range from causation by a singleinfective/transforming event to the evolution of an increasingly“disease-like” or ‘cancer-like’ tissue type leading ultimately to onewith fully pathogenic or malignant capability. Some argue that withcancer, for example, a single mutational event is sufficient to causemalignancy, while others argue that subsequent alterations are alsonecessary. Some others have suggested that increasing mutational loadand tumor grade are necessary for both initiation as well as progressionof neoplasia via a continuum of mutation-selection events at thecellular level. Some cancer targets are found only in tumor tissues,while others are present in normal tissues and are up regulated and/orover-expressed in tumor tissues. In such situations, some researchershave suggested that the over-expression is linked to the acquisition ofmalignancy, while others suggest that the over-expression is merely amarker of a trend along a path to an increasing disease state.

An ideal diagnostic and/or therapeutic antibody would be specific for anantigen present on a large number of cancers, but absent or present onlyat low levels on any normal tissue. The discovery, characterization, andisolation of a novel antigen that is specifically associated withcancer(s) would be useful in many ways. First, the antigen could be usedto make monoclonal antibodies against the antigen. An antibody wouldideally have biological activity against cancer cells and be able torecruit the immune system's response to foreign antigens. An antibodycould be administered as a therapeutic alone or in combination withcurrent treatments or used to prepare immunoconjugates linked to toxicagents. An antibody with the same specificity but with low or nobiological activity when administered alone could also be useful in thatan antibody could be used to prepare an immunoconjugate with aradio-isotope, a toxin, or a chemotherapeutic agent or liposomecontaining a chemotherapeutic agent, with the conjugated form beingbiologically active by virtue of the antibody directing the toxin to theantigen-containing cells.

One aspect desirable for an ideal diagnostic and/or therapeutic antibodyis the discovery and characterization of an antigen that is associatedwith a variety of cancers. There are few antigens that are expressed ona number of types of cancer (e.g., “pan-cancer” antigen) that havelimited expression on non-cancerous cells. The isolation andpurification of such an antigen would be useful for making antibodies(e.g., diagnostic or therapeutic) targeting the antigen. An antibodybinding to the “pan-cancer” antigen could be able to target a variety ofcancers found in different tissues in contrast to an antibody against anantigen associated with only one specific type of cancer. The antigenwould also be useful for drug discovery (e.g., small molecules) and forfurther characterization of cellular regulation, growth, anddifferentiation.

Oncostatin M (OSM) (Rose T M. Bruce A G. PNAS USA 88(19): 8641-5, 1991)is a 28 kD glycoprotein which belongs to a family of cytokinescomprising IL-6, IL-11, leukemia inhibitory factor (LIF), cililiaryneurotrophic factor (CNTF) and cardiotrophin 1 (CT-1) (Taga T. KishimotoT. Annual Review of Immunology. 15:797-819, 1997). All members share acommon signaling chain, gp130, as part of a complex family of hetero-and homodimeric receptors (Grotzinger J. et al., Proteins. 27(1):96-109,1997). OSM shares a common heterodimeric receptor with LIF, (LIFr:gp130, type I) and also has its own unique receptor comprisingOncostatin M receptor beta chain (OSM-R.beta) and gp130 (type II)(Mosley B. et al., Journal Of Biological Chemistry. 271(51):32635-32643, 1996). OSM has been known for effects on cell growth anddifferentiation (Horn D. et al, Growth Factors. 2(2-3): 157-65, 1990).OSM has also been shown to have potent, pro-inflammatory properties inmice in vivo (Modur V. et al. J. Clin Invest. 100:158-168, 1997) anddemonstrates potent synergy with IL-1 to promote articular cartilagedegradation in model systems, ex-vivo (Cawston T. Biochemical &Biophysical Research Communications. 215(1): 377-85, 1995). Antibodiesto oncostatin M, gp130, OSM-R.beta, and various complexes of thesepolypeptides are known. These antibodies are variously described asantagonists or agonists of OSM. A variety of roles have been proposed inthe scientific literature that these polypeptides may play ininflammation and the growth of tumor-derived and normal cells.

Mosley et al., U.S. Pat. No. 5,925,740, claims an antibody that isimmunoreactive with an oncostatin M receptor beta polypeptide, howeverthat specification presents no experimental data indicating that suchantibodies were actually produced and no ATCC deposit is referenced forsuch an antibody. No data showing pharmaceutical utility of the claimedantibodies was presented.

What is needed are targets on the surface of diseased and/or cancercells that may be used to diagnose and treat such diseases and/orcancers with antibodies and other agents which specifically recognizethe cell surface targets. There exists a further need, based on thediscoveries disclosed herein, for novel antibodies and other agentswhich specifically recognize targets on the surface of cells that canmodulate, either by reducing or enhancing, the disease-promotingactivities of OSM-R.beta. It is an object of this invention to identifyagonists and antagonists of human OSM-R.beta that are capable ofinhibiting its disease-associated activities. It is another object toprovide novel compounds for use in the assay of OSM-R.beta, and for useas immunogens or for selecting anti-human OSM-R.beta antibodies.

All references, publications and patent applications disclosed hereinare hereby incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

The invention provides for OSM-R.beta agonists, antagonists, modulators,and monoclonal antibodies that bind to OSM-R.beta, which is expressed ona variety of human cancers. In one aspect, the invention is a family ofmonoclonal antibodies that bind to OSM-R.beta.

In another aspect, the invention is a monoclonal antibodyanti-OSM-R.beta that is produced by the host cell line deposited on Jan.12, 2005 at the American Type Culture Collection at 10801 UniversityBoulevard, Manassas, VA 20110-2209 with a Patent Deposit Designation ofPTA-6511.

In yet another aspect, the invention is a method of generatingmonoclonal antibody anti-OSM-R.beta reactive with diseased and/orcancerous cells comprising the steps of: (a) immunizing a host mammalwith an immunogen; (b) obtaining lymphocytes from the mammal; (c) fusinglymphocytes (b) with a myeloma cell line to produce a hybridoma; (d)culturing the hybridoma of (c) to produce monoclonal antibodies; and (e)screening the antibodies to select only those antibodies which bind todiseased and/or cancerous cells or cell lines but do not bind tonon-cancerous or normal cells or cell lines, or bind to normal cells ata lower level or in a different fashion.

In another aspect, the invention is a method of generating ananti-OSM-R.beta antibody comprising culturing a host cell encoding suchantibody or progeny thereof under conditions that allow production ofthe antibody, and purifying the anti-OSM-R.beta antibody.

In another aspect, the invention provides methods of generating any ofthe antibodies (or polypeptides) described herein by expressing one ormore polynucleotides encoding the antibody (which may be separatelyexpressed as a single light or heavy chain, or both a light and a heavychain are expressed from one vector) in a suitable cell, generallyfollowed by recovering and/or isolating the antibody or polypeptides ofinterest.

In another aspect, the invention is an anti-OSM-R.beta antibody or apolypeptide (which may or may not be an antibody) that competitivelyinhibits preferential binding of an anti-OSM-R.beta antibody toOSM-R.beta. In some embodiments, the invention is an antibody or apolypeptide (which may or may not be an antibody) that bindspreferentially to the same or different epitope(s) on OSM-R.beta asother anti-OSM-R.beta antibodies.

In another aspect, the invention is an OSM-R.beta modulator (which mayor may not be a polypeptide) that competitively inhibits preferentialbinding of an anti-OSM-R.beta antibody to OSM-R.beta. In someembodiments, the invention can be a small molecule or chemical compoundthat binds preferentially to the same or different epitope(s) onOSM-R.beta as other anti-OSM-R.beta antibodies.

In yet another aspect, the invention is a composition comprisingOSM-R.beta bound by an antibody specific for an epitope of OSM-R.beta.In one embodiment, the antibody is anti-OSM-R.beta. In otherembodiments, two or more anti-OSM-R.beta antibodies are administered,with such antibodies mapping to two or more different epitopes ofOSM-R.beta. In some embodiments, the anti-OSM-R.beta antibody is linkedto a therapeutic agent or a detectable label.

In another aspect, the invention is an antibody comprising a fragment ora region of an anti-OSM-R.beta antibody. In one embodiment, the fragmentis a light chain of the antibody. In another embodiment, the fragment isa heavy chain of the antibody. In yet another embodiment, the fragmentcontains one or more variable regions from a light chain and/or a heavychain of the antibody. In yet another embodiment, the fragment containsone or more complementarity determining regions (CDRs) from a lightchain and/or a heavy chain of the antibody.

In another aspect, the invention provides polypeptides (which may or maynot be antibodies) comprising any of the following: a) one or more CDRs(or fragments thereof) from the light or heavy chain; b) three CDRs fromthe light chain; c) three CDRs from the heavy chain; d) three CDRs fromthe light chain and three CDRs from the heavy chain; e) the light chainvariable region; f) the heavy chain variable region of theanti-OSM-R.beta antibody.

In another aspect, the invention is a humanized antibody. In someembodiments, the humanized antibody comprises one or more CDRs of anon-human anti-OSM-R.beta antibody. In some embodiments, the humanizedantibody binds to the same or different epitope(s) as otheranti-OSM-R.beta antibodies. Generally, a humanized antibody of theinvention comprises one or more (one, two, three, four, five, six, orfragments thereof) CDRs which are the same and/or derived from theCDR(s) of the original non-human anti-OSM-R.beta antibody. In someembodiments, the human antibody binds to the same or differentepitope(s) as other anti-OSM-R.beta antibodies. In another aspect, theinvention is a chimeric antibody comprising variable regions derivedfrom variable regions of a heavy chain and a light chain of a non-humananti-OSM-R.beta antibody and constant regions derived from constantregions of a heavy chain and a light chain of a human antibody.

In another aspect, the invention is an isolated polynucleotide thatencodes an antibody mu-anti-OSM-R.beta that is produced by a host cellwith a deposit number of ATCC No. PTA-6511, or progeny thereof. Thisinvention encompasses antibody polypeptides having the inherent bindingor biological activities of any of the above-specified antibodies. Inanother aspect, the invention provides polynucleotides encoding any ofthe antibodies (including antibody fragments) as well as any otherpolypeptides described herein.

In another aspect, the invention is a pharmaceutical compositioncomprising any of the polypeptides (including any of the antibodiesdescribed herein) or polynucleotides described herein, such aspharmaceutical compositions comprising an anti-OSM-R.beta antibodylinked to a chemotherapeutic agent, an antibody comprising a fragment ofan anti-OSM-R.beta antibody, a humanized antibody of a non-humananti-OSM-R.beta antibody, a chimeric antibody comprising variableregions derived from variable regions of a non-human anti-OSM-R.betaantibody and constant regions derived from constant regions of a humanantibody, or a human antibody with one or more properties of a non-humananti-OSM-R.beta antibody, or any of the anti-OSM-R.beta antibodydescribed herein linked to a chemotherapeutic agent (such as aradioactive moiety), and a pharmaceutically acceptable excipient.

In one aspect, the invention is a composition comprising ananti-OSM-R.beta antibody bound to OSM-R.beta present on a diseased orcancerous cell. In preferred embodiments, the cancer cell is selectedfrom the group consisting of ovarian, lung, prostate, pancreatic, colon,and breast cancer cells. In some embodiments, the cancer cell isisolated. In some embodiments, the cancer cell is in a biologicalsample. Generally, the biological sample is from an individual, such asa human.

In another aspect, the invention is a method of diagnosing disease in anindividual by detecting OSM-R.beta on cells from the individual,particularly diseases or disorders associated with inflammatory orautoimmune responses in individuals. In other aspects of the invention,methods are provided for modulating inflammatory or autoimmune responsesin individuals. Diseases and conditions resulting from inflammation andautoimmune disorders that may be subject to treatment using thecompositions and methods of the invention include, by way ofillustration and not of limitation, multiple sclerosis, meningitis,encephalitis, stroke, other cerebral traumas, inflammatory bowel diseaseincluding ulcerative colitis and Crohn's disease, myasthenia gravis,lupus, rheumatoid arthritis, asthma, acute juvenile onset diabetes, AIDSdementia, atherosclerosis, nephritis, retinitis, atopic dermatitis,psoriasis, myocardial ischemia and acute leukocyte-mediated lung injury.

Still other indications for therapeutic use of antibodies and othertherapeutic agents of the invention include administration toindividuals at risk of organ or graft rejection. Over recent years therehas been a considerable improvement in the efficiency of surgicaltechniques for transplanting tissues and organs such as skin, kidney,liver, heart, lung, pancreas and bone marrow. Perhaps the principaloutstanding problem is the lack of satisfactory agents for inducingimmunotolerance in the recipient to the transplanted allograft or organ.When allogeneic cells or organs are transplanted into a host (i.e., thedonor and donee are different individuals from the same species), thehost immune system is likely to mount an immune response to foreignantigens in the transplant (host-versus-graft disease) leading todestruction of the transplanted tissue.

In another aspect, the invention is a method for diagnosing whether anindividual has cancer, comprising determining whether there isexpression of OSM-R.beta on selected cells from the individual, whereinthe expression of OSM-R.beta on said cells is indicative of said cancer.In some embodiments, the expression of OSM-R.beta is determined using ananti-OSM-R.beta antibody. In some embodiments, the method involvesdetecting the level of OSM-R.beta expression from cells. The term“detection” as used herein includes qualitative and/or quantitativedetection (measuring levels) with or without reference to a control.

In yet another aspect, the invention is a method of diagnosing cancer inan individual by detecting OSM-R.beta on or released from cells from theindividual, wherein the cancer is selected from the group including butnot limited to adrenal gland tumors, AIDS-associated cancers, alveolarsoft part sarcoma, astrocytic tumors, bladder cancer (squamous cellcarcinoma and transitional cell carcinoma), bone cancer (adamantinoma,aneurismal bone cysts, osteochondroma, osteosarcoma), brain and spinalcord cancers, metastatic brain tumors, breast cancer, carotid bodytumors, cervical cancer, chondrosarcoma, dhordoma, chromophobe renalcell carcinoma, clear cell carcinoma, colon cancer, colorectal cancer,cutaneous benign fibrous histiocytomas, desmoplastic small round celltumors, ependymomas, Ewing's tumors, extraskeletal myxoidchondrosarcoma, fibrogenesis imperfecta ossium, fibrous dysplasia of thebone, gallbladder and bile duct cancers, gestational trophoblasticdisease, germ cell tumors, head and neck cancers, islet cell tumors,Kaposi's Sarcoma, kidney cancer (nephroblastoma, papillary renal cellcarcinoma), leukemias, lipoma/benign lipomatous tumors,liposarcoma/malignant lipomatous tumors, liver cancer (hepatoblastoma,hepatocellular carcinoma), lymphomas, lung cancers (small cellcarcinoma, adenocarcinoma, squamous cell carcinoma, large cell carcinomaetc.), medulloblastoma, melanoma, meningiomas, multiple endocrineneoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma,glioblastoma, neuroendocrine tumors, ovarian cancer, pancreatic cancers,papillary thyroid carcinomas, parathyroid tumors, pediatric cancers,peripheral nerve sheath tumors, phaeochromocytoma, pituitary tumors,prostate cancer, posterious unveal melanoma, rare hematologic disorders,renal metastatic cancer, rhabdoid tumor, rhabdomysarcoma, sarcomas, skincancer, soft-tissue sarcomas, squamous cell cancer, stomach cancer,synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, thyroidmetastatic cancer, and uterine cancers (carcinoma of the cervix,endometrial carcinoma, and leiomyoma). In some situations, theantibodies of this invention are not used to treat Kaposi's sarcoma.

In another aspect, the invention is a method for aiding diagnosis ofcancer (such as but not limited to ovarian, lung, prostate, pancreatic,colon, or breast cancer) in an individual comprising determining theexpression of OSM-R.beta in a biological sample from the individual. Insome embodiments, the expression of OSM-R.beta is determined using ananti-OSM-R.beta antibody. In some embodiments, the method is detectingthe level of OSM-R.beta expression from cells. The OSM-R.beta releasedfrom the cancer may contribute to elevated levels of OSM-R.beta or aportion thereof, being detectable in body fluids (e.g., blood, salivaryor gut mucinous secretions).

In yet another aspect, the invention is a method of treating cancer byadministering an effective amount of an antibody that binds toOSM-R.beta sufficient to reduce growth of cancerous cells. In someembodiments, the antibody is an anti-OSM-R.beta antibody. In certainembodiments, the cancerous cells are selected from the group includingbut not limited to adrenal gland tumors, AIDS-associated cancers,alveolar soft part sarcoma, astrocytic tumors, bladder cancer (squamouscell carcinoma and transitional cell carcinoma), bone cancer(adamantinoma, aneurismal bone cysts, osteochondroma, osteosarcoma),brain and spinal cord cancers, metastatic brain tumors, breast cancer,carotid body tumors, cervical cancer, chondrosarcoma, dhordoma,chromophobe renal cell carcinoma, clear cell carcinoma, colon cancer,colorectal cancer, cutaneous benign fibrous histiocytomas, desmoplasticsmall round cell tumors, ependymomas, Ewing's tumors, extraskeletalmyxoid chondrosarcoma, fibrogenesis imperfecta ossium, fibrous dysplasiaof the bone, gallbladder and bile duct cancers, gestationaltrophoblastic disease, germ cell tumors, head and neck cancers, isletcell tumors, Kaposi's Sarcoma, kidney cancer (nephroblastoma, papillaryrenal cell carcinoma), leukemias, lipoma/benign lipomatous tumors,liposarcoma/malignant lipomatous tumors, liver cancer (hepatoblastoma,hepatocellular carcinoma), lymphomas, lung cancers (small cellcarcinoma, adenocarcinoma, squamous cell carcinoma, large cell carcinomaetc.), medulloblastoma, melanoma, meningiomas, multiple endocrineneoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma,glioblastoma, neuroendocrine tumors, ovarian cancer, pancreatic cancers,papillary thyroid carcinomas, parathyroid tumors, pediatric cancers,peripheral nerve sheath tumors, phaeochromocytoma, pituitary tumors,prostate cancer, posterious unveal melanoma, rare hematologic disorders,renal metastatic cancer, rhabdoid tumor, rhabdomysarcoma, sarcomas, skincancer, soft-tissue sarcomas, squamous cell cancer, stomach cancer,synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, thyroidmetastatic cancer, and uterine cancers (carcinoma of the cervix,endometrial carcinoma, and leiomyoma). In certain preferred embodiments,the cancerous cells are selected from the group of solid tumorsincluding but not limited to breast cancer, colon cancer, prostatecancer, lung cancers, sarcoma, renal metastatic cancer, thyroidmetastatic cancer, and clear cell carcinoma.

In yet another aspect, the invention is a method of delaying orpreventing development of metastasis in an individual who has or has hada primary tumor, comprising administering an effective amount of anOSM-R.beta modulator. This modulator may be given alone or inconjunction with other therapies, such as radiation, chemotherapy, orsurgery. In certain preferred embodiments, the primary tumor has beensubjected to at least one round of surgery, radiation, and/orchemotherapy before administration of the OSM-R.beta modulator. In otherembodiments, the OSM-R.beta modulator is administered prior to orconcurrent with treatment of the individual with surgery, radiation,and/or chemotherapy. In certain particularly preferred embodiments, thatOSM-R.beta modulator is at least one anti-OSM-R.beta antibody. Inanother aspect, the invention is a method of inhibiting growth and/orproliferation of cancer cells in vitro or in an individual comprisingadministering an effective amount of a composition comprising ananti-OSM-R.beta antibody associated with (including linked to) achemotherapeutic agent to the cell culture or sample, or to theindividual.

In yet another aspect, the invention is a method of delivering atherapeutic agent to a cancerous cell in an individual by administeringto the individual an effective amount of at least one member of a familyof antibodies, which bind specifically to OSM-R.beta. In otherembodiments, an anti-OSM-R.beta antibody is delivered to an individualin combination with (including linked to) another therapeutic agent.

In particularly preferred embodiments, the OSM-R.beta modulators of thisinvention are used in the diagnosis, prevention or treatment ofpancreatic, breast, ovarian, kidney and lung carcinomas, melanomas,neuroblastomas and glioblastomas.

In some embodiments, the anti-OSM-R.beta antibody is a humanizedantibody derived from a named antibody herein (generally, but notnecessarily, comprising one or more partial or intact CDRs of theantibody). In some embodiments, the anti-OSM-R.beta antibody is a humanantibody with one or more properties of the named antibody. In someembodiments, the chemotherapeutic agent (such as a toxin or aradioactive molecule) is delivered into the cancer cells (isinternalized). In some embodiments, the agent is saporin.

In another aspect, the invention is a method of treating cancer in anindividual comprising administering an effective amount of a compositioncomprising an anti-OSM-R.beta antibody associated with (including linkedto) a chemotherapeutic agent to the individual.

The present invention further provides methods for modulating, either byenhancing or reducing, the association of OSM-R.beta with a cytoplasmicsignaling partner. The association of OSM-R.beta with a cytoplasmicsignaling partner can be impacted by contacting an OSM-R.beta moleculepresenting on a cell surface, with an agent that modulates the bindingof the signaling partner to OSM-R.beta. Agents which block or reduceOSM-R.beta association with its binding and/or signaling partners can beused to modulate biological and pathological processes which areinvolved in OSM-R.beta-mediated inflammation or immune responses.Pathological processes involving this action include tumor-associatedcell growth.

Agents can be tested for their ability to block, reduce, enhance orotherwise modulate the association of OSM-R.beta with a binding partner,such as an anti-OSM-R.beta antibody. Specifically, an agent can betested for the ability to modulate such an interaction by incubating apeptide comprising the OSM-R.beta interaction site (typically in itsnative conformation as it exists on intact living cells) with a bindingpartner and a test agent, and determining whether the test agent reducesor enhances the binding of the binding partner to the OSM-R.betapeptide.

Agonists, antagonists, and other modulators of OSM-R.beta function areexpressly included within the scope of this invention. These agonists,antagonists and modulators are polypeptides that comprise one or more ofthe antigenic determinant sites in OSM-R.beta, or comprise one or morefragments of such sites, variants of such sites, or peptidomimetics ofsuch sites. These agonistic, antagonistic, and OSM-R.beta modulatorycompounds are provided in linear or cyclized form, and optionallycomprise at least one amino acid residue that is not commonly found innature or at least one amide isostere. These compounds may beglycosylated. The agonists, antagonists, and other modulators ofOSM-R.beta function of this invention are desirably used in all of theembodiments and methods described above with reference to antibodies.

In certain aspects, the invention is a method for aiding in thediagnosis of disease in an individual comprising the steps of (i)assaying for the presence of OSM-R.beta in a blood or tissue sampleobtained from an individual; (ii) detecting whether said sample has anincreased amount of a OSM-R.beta marker relative to a normal(non-diseased) blood or tissue sample; and (iii) correlating anincreased amount of said marker to a positive diagnosis or correlatingthe absence of an increased amount of said marker to a negativediagnosis for disease. In certain embodiments, the marker is detectedusing an anti-OSM-R.beta antibody. In certain embodiments, the method iseffected by a technique selected from the group consisting ofradionuclide imaging, flow cytometry, and immunohistochemistry.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the graphed results of several experiments illustrating thein vitro inhibition of a human renal carcinoma cell line grown in amonolayer by an anti-OSM-R.beta antibody.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides an antigen, OSM-R.beta, which is expressed oncancerous cells of various tissue types, including but not limited tobreast, colon, lung, and prostate cancers. Further, the inventionprovides monoclonal antibodies and polypeptides that bind to OSM-R.betaand methods making and using these antibodies and polypeptides todiagnose and treat various diseases human cancers associated withexpression and/or over-expression of OSM-R.beta.

I. General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, Molecular Cloning: ALaboratory Manual, second edition (Sambrook et al., 1989) Cold SpringHarbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methodsin Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook(J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I.Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J.P.Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture:Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell,eds., 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press,Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C.Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M.Miller and M. P. Calos, eds., 1987); Current Protocols in MolecularBiology (F. M. Ausubel et al., eds., 1987); PCR: The Polymerase ChainReaction, (Mullis et al., eds., 1994); Current Protocols in Immunology(J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology(Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers,1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D.Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practicalapproach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000);Using antibodies: a laboratory manual (E. Harlow and D. Lane (ColdSpring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer:Principles and Practice of Oncology (V. T. DeVita et al., eds., J. B.Lippincott Company, 1993).

II. Definitions

“OSM-R.beta” refers to that novel polypeptide antigen with aglycosylated molecular weight of approximately 130 kD to 180 kD, againstwhich the antibodies of the present invention are directed. TheOSM-R.beta antigen is a cell surface glycoprotein bound by OSM-R.betaand present on kidney and several types of carcinomas. This antigen mayhave more than one different epitope, and some embodiments of thisinvention comprise OSM-R.beta modulators that are directed against oneof two or more specific epitopes of the OSM-R.beta antigen. It iscurrently believed that OSM-R.beta may be over-expressed in certaincancer cells in comparison to their normal tissue counterparts.

Agonists, antagonists, and other modulators of OSM-R.beta function areexpressly included within the scope of this invention. These agonists,antagonists and modulators are polypeptides that comprise one or more ofthe antigenic determinant sites in OSM-R.beta, or comprise one or morefragments of such sites, variants of such sites, or peptidomimetics ofsuch sites. These agonistic, antagonistic, and OSM-R.beta modulatorycompounds are provided in linear or cyclized form, and optionallycomprise at least one amino acid residue that is not commonly found innature or at least one amide isostere. These compounds may beglycosylated.

More specifically, the terms “OSM-R.beta agonist”, “antagonist” or“modulator” as used herein are defined as any compound that (1) iscapable of disrupting or blocking the interaction between humanOSM-R.beta and its native ligands or an anti-OSM-R.beta antibody; (2) iscapable of binding to human OSM-R.beta and its native ligands or ananti-OSM-R.beta antibody; (3) contains an antigenic site that can beused in the raising of antibodies capable of binding to human OSM-R.betaand its native ligands or an anti-OSM-R.beta antibody; (4) contains anantigenic site that can be used in the screening of antibodies capableof binding to human OSM-R.beta and its native ligands or ananti-OSM-R.beta antibody; (5) contains an antigenic site that an be usedin the raising of antibodies capable of disrupting or blocking theinteraction between human OSM-R.beta and its native ligands or ananti-OSM-R.beta antibody; (6) contains an antigenic site that can beused in the screening of antibodies capable of disrupting or blockingthe interaction between human OSM-R.beta and its native ligands or ananti-OSM-R.beta antibody. OSM-R.beta modulators may be “OSM-R.betaagonists” or “OSM-R.beta antagonists” depending on whether theiractivity enhances or inhibits normal OSM-R.beta biological activity,respectively.

OSM-R.beta agonists, antagonists and modulators include OSM-R.betavariants, OSM-R.beta peptide antagonists, peptidomimetics, and smallmolecules, anti-OSM-R.beta antibodies and immunoglobulin variants, aminoacid variants of human OSM-R.beta including amino acid substitution,deletion, and addition variants, or any combination thereof, andchimeric immunoglobulins. The OSM-R.beta agonists, antagonists andmodulators of this invention are based on the inventors' identificationof the OSM-R.beta domains involved in the binding of human OSM-R.beta toits native ligands or anti-OSM-R.beta antibodies. Thus, the inventionprovides OSM-R.beta agonists, antagonists and modulators with molecularstructures that duplicate or mimic one or more of the anti-OSM-R.betabinding domains of human OSM-R.beta.

As used herein, the term “OSM-R.beta variant” denotes any amino acidvariant of human OSM-R.beta, including amino acid substitution,deletion, and addition variants, or any combination thereof. Thedefinition encompasses chimeric molecules such as humanOSM-R.beta/non-human chimeras and other hybrid molecules. Also includedin the definition is any fragment of an OSM-R.beta variant molecule thatcomprises the variant or hybrid region(s) of the molecule.

An “antibody” is an immunoglobulin molecule capable of specific bindingto a target, such as a carbohydrate, polynucleotide, lipid, polypeptide,etc., through at least one antigen recognition site, located in thevariable region of the immunoglobulin molecule. As used herein, the termencompasses not only intact polyclonal or monoclonal antibodies, butalso fragments thereof (such as Fab, Fab′, F(ab′)₂, Fv), single chain(ScFv), mutants thereof, naturally occurring variants, fusion proteinscomprising an antibody portion with an antigen recognition site of therequired specificity, humanized antibodies, chimeric antibodies, and anyother modified configuration of the immunoglobulin molecule thatcomprises an antigen recognition site of the required specificity.

A “monoclonal antibody” refers to a homogeneous antibody populationwherein the monoclonal antibody is comprised of amino acids (naturallyoccurring and non-naturally occurring) that are involved in theselective binding of an antigen. Monoclonal antibodies are highlyspecific, being directed against a single antigenic site. The term“monoclonal antibody” encompasses not only intact monoclonal antibodiesand full-length monoclonal antibodies, but also fragments thereof (suchas Fab, Fab′, F(ab′)₂, Fv), single chain (ScFv), mutants thereof, fusionproteins comprising an antibody portion, humanized monoclonalantibodies, chimeric monoclonal antibodies, and any other modifiedconfiguration of the immunoglobulin molecule that comprises an antigenrecognition site of the required specificity and the ability to bind toan antigen. It is not intended to be limited as regards to the source ofthe antibody or the manner in which it is made (e.g., by hybridoma,phage selection, recombinant expression, transgenic animals, etc.). Theterm includes whole immunoglobulins as well as the fragments etc.described above under the definition of “antibody”.

“Humanized” antibodies refer to a chimeric molecule, generally preparedusing recombinant techniques, having an antigen binding site derivedfrom an immunoglobulin from a non-human species and the remainingimmunoglobulin structure of the molecule based upon the structure and/orsequence of a human immunoglobulin. The antigen-binding site maycomprise either complete variable domains fused onto constant domains oronly the complementarity determining regions (CDRs) grafted ontoappropriate framework regions in the variable domains. Antigen bindingsites may be wild type or modified by one or more amino acidsubstitutions. This eliminates the constant region as an immunogen inhuman individuals, but the possibility of an immune response to theforeign variable region remains (LoBuglio, A. F. et al., (1989) ProcNatl Acad Sci USA 86:4220-4224). Another approach focuses not only onproviding human-derived constant regions, but modifying the variableregions as well so as to reshape them as closely as possible to humanform. It is known that the variable regions of both heavy and lightchains contain three complementarity-determining regions (CDRs) whichvary in response to the antigens in question and determine bindingcapability, flanked by four framework regions (FRs) which are relativelyconserved in a given species and which putatively provide a scaffoldingfor the CDRs. When nonhuman antibodies are prepared with respect to aparticular antigen, the variable regions can be “reshaped” or“humanized” by grafting CDRs derived from nonhuman antibody on the FRspresent in the human antibody to be modified. Application of thisapproach to various antibodies has been reported by Sato, K., et al.,(1993) Cancer Res 53:851-856. Riechmann, L., et al., (1988) Nature332:323-327; Verhoeyen, M., et al., (1988) Science 239:1534-1536;Kettleborough, C. A., et al., (1991) Protein Engineering 4:773-3783;Maeda, H., et al., (1991) Human Antibodies Hybridoma 2:124-134; Gorman,S. D., et al., (1991) Proc Natl Acad Sci USA 88:4181-4185; Tempest, P.R., et al., (1991) Bio/Technology 9:266-271; Co, M. S., et al., (1991)Proc Natl Acad Sci USA 88:2869-2873; Carter, P., et al., (1992) ProcNatl Acad Sci USA 89:4285-4289; and Co, M. S. et al., (1992) J Immunol148:1149-1154. In some embodiments, humanized antibodies preserve allCDR sequences (for example, a humanized mouse antibody which containsall six CDRs from the mouse antibodies). In other embodiments, humanizedantibodies have one or more CDRs (one, two, three, four, five, six)which are altered with respect to the original antibody, which are alsotermed one or more CDRs “derived from” one or more CDRs from theoriginal antibody.

An epitope that “specifically binds” or “preferentially binds” (usedinterchangeably herein) to an antibody or a polypeptide is a term wellunderstood in the art, and methods to determine such specific orpreferential binding are also well known in the art. A molecule is saidto exhibit “specific binding” or “preferential binding” if it reacts orassociates more frequently, more rapidly, with greater duration and/orwith greater affinity with a particular cell or substance than it doeswith alternative cells or substances. An antibody “specifically binds”or “preferentially binds” to a target if it binds with greater affinity,avidity, more readily, and/or with greater duration than it binds toother substances. For example, an antibody that specifically orpreferentially binds to an OSM-R.beta epitope is an antibody that bindsthis OSM-R.beta epitope with greater affinity, avidity, more readily,and/or with greater duration than it binds to other OSM-R.beta epitopesor non-OSM-R.beta epitopes. It is also understood by reading thisdefinition that, for example, an antibody (or moiety or epitope) thatspecifically or preferentially binds to a first target may or may notspecifically or preferentially bind to a second target. As such,“specific binding” or “preferential binding” does not necessarilyrequire (although it can include) exclusive binding. Generally, but notnecessarily, reference to binding means preferential binding.

The term “immunologically active” in reference to an epitope being or“remaining immunologically active” refers to the ability of an antibody(e.g., anti-OSM-R.beta antibody) to bind to the epitope under differentconditions, for example, after the epitope has been subjected toreducing and denaturing conditions.

Different biological functions are associated with anti-OSM-R.betaantibodies, including, but not limited to, ability to bind to OSM-R.beta(including OSM-R.beta on cancer cells, including but not limited toovarian, prostate, pancreatic, lung, colon, or breast cancer cells);ability to bind to a portion of OSM-R.beta that is exposed on thesurface of a living cell in vitro or in vivo; ability to deliver achemotherapeutic agent to cancerous cells (such as ovarian, prostate,pancreatic, lung, colon, or breast cancer cells) expressing OSM-R.beta;ability to deliver a therapeutic agent or detectable marker into cancercells expressing OSM-R.beta. As discussed herein, polypeptides(including antibodies) of the invention may have any one or more ofthese characteristics.

An “anti-OSM-R.beta equivalent antibody” or “anti-OSM-R.beta equivalentpolypeptide” refers to an antibody or a polypeptide having one or morebiological functions associated with an anti-OSM-R.beta antibody, suchas, for example binding specificity.

As used herein, “agent” refers to a biological, pharmaceutical, orchemical compound. Non-limiting examples include simple or complexorganic or inorganic molecule, a peptide, a protein, an oligonucleotide,an antibody, an antibody derivative, antibody fragment, a vitaminderivative, a carbohydrate, a toxin, or a chemotherapeutic compound.Various compounds can be synthesized, for example, small molecules andoligomers (e.g., oligopeptides and oligonucleotides), and syntheticorganic compounds based on various core structures. In addition, variousnatural sources can provide compounds for screening, such as plant oranimal extracts, and the like. A skilled artisan can readily recognizethat there is no limit as to the structural nature of the agents of thepresent invention.

Agents that are employed in the methods of this invention can berandomly selected or rationally selected or designed. As used herein, anagent is said to be randomly selected when the agent is chosen randomlywithout considering the specific sequences involved in the associationof OSM-R.beta with its native binding partners or known antibodies. Anexample of randomly selected agents is the use of a chemical library ora peptide combinatorial library.

As used herein, an agent is said to be rationally selected or designedwhen the agent is chosen on a nonrandom basis that takes into accountthe sequence of the target site and/or its conformation in connectionwith the agent's action. With respect to anti-OSM-R.beta agents, it iscurrently believed that there are at least three epitopes on OSM-R.betaagainst which antibodies can be raised and therefore at least threesites of action for agents that block OSM-R.beta/anti-OSM-R.betainteraction. This invention also encompasses agents that act at thesites of interaction between OSM-R.beta and its native binding partner,although other ligands and their active OSM-R.beta-interactive sites arealso encompassed within the scope of this invention, whether currentlyknown or later identified. Agents can be rationally selected orrationally designed by utilizing the peptide sequences that make up thecontact sites of the receptor/ligand and/or OSM-R.beta/anti-OSM-R.betaantibody complex. For example, a rationally selected peptide agent canbe a peptide whose amino acid sequence is identical to an epitopeappearing on OSM-R.beta as it is exposed on the surface of a living cellin its native environment. Such an agent will reduce or block theassociation of the anti-OSM-R.beta antibody with OSM-R.beta, or theassociation of OSM-R.beta with its native ligand, as desired, by bindingto the anti-OSM-R.beta antibody or to the native ligand.

As used herein, the term “labeled”, with regard to the antibody, isintended to encompass direct labeling of the antibody by coupling (i.e.,physically linking) a detectable substance, such as a radioactive agentor a fluorophore (e.g. fluorescein isothiocyanate (FITC) orphycoerythrin (PE)) to the antibody, as well as indirect labeling of theprobe or antibody by reactivity with a detectable substance.

As used herein, the term “association”, with regard to the antibody,includes covalent and non-covalent attachment or binding to an agent(e.g., chemotherapeutic agent). The antibody can be associated with anagent (e.g., chemotherapeutic agent) by direct binding or indirectbinding via attachment to a common platform, such that the antibodydirects the localization of the agent to the cancerous cell to which theantibody binds and wherein the antibody and agent do not substantiallydissociate under physiological conditions such that the agent is nottargeted to the same cancerous cell to which the antibody binds or suchthat the agent's potency is not decreased.

A “biological sample” encompasses a variety of sample types obtainedfrom an individual and can be used in a diagnostic or monitoring assay.The definition encompasses saliva, blood and other liquid samples ofbiological origin, solid tissue samples such as a biopsy specimen ortissue cultures or cells derived therefrom, and the progeny thereof, forexample, cells obtained from a tissue sample collected from anindividual suspected of having cancer, in preferred embodiments fromovary, lung, prostate, pancreas, colon, and breast tissue. Thedefinition also includes samples that have been manipulated in any wayafter their procurement, such as by treatment with reagents,solubilization, or enrichment for certain components, such as proteinsor polynucleotides, or embedding in a semi-solid or solid matrix forsectioning purposes. The term “biological sample” encompasses a clinicalsample, and also includes cells in culture, cell supernatants, celllysates, serum, plasma, biological fluid, and tissue samples.

A “host cell” includes an individual cell or cell culture that can be orhas been a recipient for vector(s) for incorporation of polynucleotideinserts. Host cells include progeny of a single host cell, and theprogeny may not necessarily be completely identical (in morphology or ingenomic DNA complement) to the original parent cell due to natural,accidental, or deliberate mutation. A host cell includes cellstransfected in vivo with a polynucleotide(s) of this invention.

As used herein, “preventing or delaying development of metastasis” meansto stop, defer, hinder, slow, retard, stabilize, and/or postponedevelopment of metastasis. This delay can be of varying lengths of time,depending on the history of the cancer and/or individual being treated.As is evident to one skilled in the art, a sufficient or significantdelay can, in effect, encompass prevention, in that the individual doesnot develop the metastasis.

An “effective amount” of a pharmaceutical composition, in oneembodiment, is an amount sufficient to effect beneficial or desiredresults including, without limitation, clinical results such asshrinking the size of the tumor (in the cancer context, for example,breast or prostate cancer), retardation of cancerous cell growth,preventing or delaying the development of metastasis, decreasingsymptoms resulting from the disease, increasing the quality of life ofthose suffering from the disease, decreasing the dose of othermedications required to treat the disease, enhancing the effect ofanother medication such as via targeting and/or internalization,delaying the progression of the disease, and/or prolonging survival ofindividuals. An effective amount can be administered in one or moreadministrations. For purposes of this invention, an effective amount ofdrug, compound, or pharmaceutical composition is an amount sufficient toreduce the proliferation of (or destroy) cancerous cells and to reduceand/or delay the development, or growth, of metastases of cancerouscells, either directly or indirectly. In some embodiments, an effectiveamount of a drug, compound, or pharmaceutical composition may or may notbe achieved in conjunction with another drug, compound, orpharmaceutical composition. Thus, an “effective amount” may beconsidered in the context of administering one or more chemotherapeuticagents, and a single agent may be considered to be given in an effectiveamount if, in conjunction with one or more other agents, a desirableresult may be or is achieved. While individual needs vary, determinationof optimal ranges of effective amounts of each component is within theskill of the art. Typical dosages comprise 0.1-to 100 mg/kg/body weight.The preferred dosages comprise 1-to 100-mg/kg/body weight. Preferreddosages comprise 10 to 20-mg/kg/body weight and 10-to 100-mg/kg/bodyweight, and the most preferred dosages comprise approximately 1-to10-mg/kg/body weight.

As used herein, a nucleic acid molecule or agent, antibody, compositionor cell, etc., is said to be “isolated” when that nucleic acid molecule,agent, antibody, composition, or cell, etc. is substantially separatedfrom contaminant nucleic acid molecules, antibodies, agents,compositions, or cells, etc. from its original source.

An “individual” is a vertebrate, preferably a mammal, more preferably ahuman. Mammals include, but are not limited to, farm animals, sportanimals, pets, primates, mice and rats.

The terms “polypeptide”, “oligopeptide”, “peptide” and “protein” areused interchangeably herein to refer to polymers of amino acids of anylength. The polymer may be linear or branched, it may comprise modifiedamino acids, and it may be interrupted by non-amino acids. The termsalso encompass an amino acid polymer that has been modified naturally orby intervention; for example, disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling component. Alsoincluded within the definition are, for example, polypeptides containingone or more analogs of an amino acid (including, for example, unnaturalamino acids, etc.), as well as other modifications known in the art. Itis understood that, because the polypeptides of this invention are basedupon an antibody, the polypeptides can occur as single chains orassociated chains.

A “variable region” of an antibody refers to the variable region of theantibody light chain or the variable region of the antibody heavy chain,either alone or in combination.

A “constant region” of an antibody refers to the constant region of theantibody light chain or the constant region of the antibody heavy chain,either alone or in combination.

Also encompassed within the scope of the invention are peptidomimeticsof the OSM-R.beta peptide agonists, antagonists and modulators(including anti-OSM-R.beta antibodies) described herein. Suchpeptidomimetics include peptides wherein at least one amino acid residueis substituted with an amino acid residue that is not commonly found innature, such as the D isomer of the amino acid or an N-alkylated speciesof the amino acid. In other embodiments, peptidomimetics are constructedby replacing at least one amide bond (—C(.dbd.O)—NH—) in a OSM-R.betapeptide agonist, antagonist or modulators with an amide isostere.Suitable amide isosteres include —CH.sub.2—NH—, —CH.sub.2—S—,—CH.sub.2—S(O).sub.n—(where n is 1 or 2), —CH.sub.2—CH.sub.2—,—CH.dbd.CH—(E or Z), —C(.dbd.O)—CH.sub.2—, —CH(CN)—NH—,—C(OH)—CH.sub.2—, and —O—C(.dbd.O)—NH—. The amide bonds in a OSM-R.betapeptide agonist, antagonist or modulator that are suitable candidatesfor replacement with amide isosteres include bonds that are hydrolyzableby the endogenous esterases or proteases of the intended subject ofOSM-R.beta peptide agonist, antagonist or modulator treatment.

As used herein, “substantially pure” refers to material that is at least50% pure (i.e., free from contaminants), more preferably at least 90%pure, more preferably at least 95% pure, more preferably at least 98%pure, more preferably at least 99% pure, or greater, pure.

“Toxin” refers to any substance, which effects an adverse responsewithin a cell. For example, a toxin directed to a cancerous cell wouldhave an adverse, sometimes deleterious effect, on the cancerous cell.Examples of toxins include, but are not limited to, radioisotopes,calicheamicin, and maytansinoids.

As used herein, “treatment” or “treating” is an approach for obtainingbeneficial or desired results including and preferably clinical results.For purposes of this invention, beneficial or desired clinical resultsinclude, but are not limited to, one or more of the following: reducingthe proliferation of (or destroying) cancerous cells or other diseased,reducing metastasis of cancerous cells found in cancers, shrinking thesize of the tumor, decreasing symptoms resulting from the disease,increasing the quality of life of those suffering from the disease,decreasing the dose of other medications required to treat the disease,delaying the progression of the disease, and/or prolonging survival ofindividuals.

As used herein, a “therapeutic agent” means any agent useful for therapy(here, generally in the cancer context) including anti-tumor drugs,toxins or cytotoxins, cytotoxin agents, and radioactive agents.

“Active immune response” refers to the development and on-goingproduction of antibodies in vivo directed against an antigen, inresponse to the administration of the antigen, or DNA vectors coding forthat antigen, to the host mammal by intravenous, intramuscular,subcutaneous, or other mode of administration with or without anadjuvant. Active immune response can also include other aspects of theimmune response, such as a cellular immune response.

III. Methods of Making Antibodies and Polypeptides

Methods of making monoclonal antibodies are known in the art. One methodwhich may be employed is the method of Kohler and Milstein, Nature256:495-497 (1975) or a modification thereof. Typically, monoclonalantibodies are developed in non-human species, such as mice. In general,a mouse or rat is used for immunization but other animals may also beused. The antibodies are produced by immunizing mice with an immunogenicamount of cells, cell extracts, or protein preparations that containhuman OSM-R.beta. The immunogen can be, but is not limited to, primarycells, cultured cell lines, cancerous cells, nucleic acids, or tissue.In one embodiment, human lung cancer cells are used. In anotherembodiment, human normal tissue progenitor cells are used. Methods forisolating and culturing human such cells are detailed in Example 1.Cells used for immunization, for example, human lung cancer, kidney,ovarian cells or human progenitor cells, may be cultured for a period oftime (at least 24 hours) prior to their use as an immunogen. Cells maybe used as immunogens by themselves or in combination with anon-denaturing adjuvant, such as Ribi. In general, cells should be keptintact and preferably viable when used as immunogens. Intact cells mayallow antigens to be better detected than ruptured cells by theimmunized animal. Use of denaturing or harsh adjuvants, e.g., Freud'sadjuvant, may rupture the human fetal kidney or other cells andtherefore is discouraged. The immunogen may be administered multipletimes at periodic intervals such as, bi-weekly, or weekly, or may beadministered in such a way as to maintain viability in the animal (e.g.,in a tissue recombinant). Example 2 describes methods used to generateanti-OSM-R.beta antibodies and may be used to generate other monoclonalantibodies, which bind to OSM-R.beta.

In one embodiment, monoclonal antibodies, which bind to OSM-R.beta areobtained by using host cells that over-express OSM-R.beta as animmunogen. Such cells include, by way of example and not by limitation,human fetal kidney cells and human colon cancer cells.

To monitor the antibody response, a small biological sample (e.g.,blood) may be obtained from the animal and tested for antibody titeragainst the immunogen. The spleen and/or several large lymph nodes canbe removed and dissociated into single cells. If desired, the spleencells may be screened (after removal of non-specifically adherent cells)by applying a cell suspension to a plate or to a well coated with theantigen. B-cells, expressing membrane-bound immunoglobulin specific forthe antigen, will bind to the plate, and are not rinsed away with therest of the suspension. Resulting B-cells, or all dissociated spleencells, can then be fused with myeloma cells (e.g., X63-Ag8.653 and thosefrom the Salk Institute, Cell Distribution Center, San Diego, Calif.).Polyethylene glycol (PEG) may be used to fuse spleen or lymphocytes withmyeloma cells to form a hybridoma. The hybridoma is then cultured in aselective medium (e.g., hypoxanthine, aminopterin, thymidine medium,otherwise known as “HAT medium”). The resulting hybridomas are thenplated by limiting dilution, and are assayed for the production ofantibodies that bind specifically to the immunogen (e.g., surface of thehuman fetal kidney cells, surface of cancer cell lines, Ag-OSM-R.beta,fetal bladder sections, etc.) using FACS or immunohistochemistry (IHCscreening). The selected monoclonal antibody-secreting hybridomas arethen cultured either in vitro (e.g., in tissue culture bottles or hollowfiber reactors), or in vivo (e.g., as ascites in mice). Example 3provides further details about the methods utilized to obtain and screenan anti-OSM-R.beta antibody.

As another alternative to the cell fusion technique, EBV immortalized Bcells may be used to produce monoclonal antibodies of the subjectinvention. The hybridomas are expanded and subcloned, if desired, andsupernatants are assayed for anti-immunogen activity by conventionalassay procedures (e.g., FACS, IHC, radioimmunoassay, enzyme immunoassay,fluorescence immunoassay, etc.).

In another alternative, monoclonal antibody anti-OSM-R.beta and anyother equivalent antibodies can be sequenced and produced recombinantlyby any means known in the art (e.g., humanization, use of transgenicmice to produce fully human antibodies, phage display technology, etc.).In one embodiment, anti-OSM-R.beta monoclonal antibody is sequenced andthe polynucleotide sequence is then cloned into a vector for expressionor propagation. The sequence encoding the antibody of interest may bemaintained in a vector in a host cell and the host cell can then beexpanded and frozen for future use.

The polynucleotide sequence of monoclonal antibody anti-OSM-R.beta andany other equivalent antibodies may be used for genetic manipulation togenerate a “humanized” antibody, to improve the affinity, or othercharacteristics of the antibody. The general principle in humanizing anantibody involves retaining the basic sequence of the antigen-bindingportion of the antibody, while swapping the non-human remainder of theantibody with human antibody sequences. There are four general steps tohumanize a monoclonal antibody. These are: (1) determining thenucleotide and predicted amino acid sequence of the starting antibodylight and heavy variable domains (2) designing the humanized antibody,i.e., deciding which antibody framework region to use during thehumanizing process (3) the actual humanizing methodologies/techniquesand (4) the transfection and expression of the humanized antibody. See,for example, U.S. Pat. Nos. 4,816,567; 5,807,715; 5,866,692; and6,331,415.

A number of “humanized” antibody molecules comprising an antigen-bindingsite derived from a non-human immunoglobulin have been described,including chimeric antibodies having rodent or modified rodent V regionsand their associated complementarity determining regions (CDRs) fused tohuman constant domains. See, for example, Winter et al. Nature349:293-299 (1991), Lobuglio et al. Proc. Nat. Acad. Sci. USA86:4220-4224 (1989), Shaw et al. J. Immunol. 138:4534-4538 (1987), andBrown et al. Cancer Res. 47:3577-3583 (1987). Other references describerodent CDRs grafted into a human supporting framework region (FR) priorto fusion with an appropriate human antibody constant domain. See, forexample, Riechmann et al. Nature 332:323-327 (1988), Verhoeyen et al.Science 239:1534-1536 (1988), and Jones et al. Nature 321:522-525(1986). Another reference describes rodent CDRs supported byrecombinantly veneered rodent framework regions. See, for example,European Patent Publication No. 519,596. These “humanized” molecules aredesigned to minimize unwanted immunological response toward rodentanti-human antibody molecules, which limits the duration andeffectiveness of therapeutic applications of those moieties in humanrecipients. Other methods of humanizing antibodies that may also beutilized are disclosed by Daugherty et al., Nucl. Acids Res.,19:2471-2476 (1991) and in U.S. Pat. Nos. 6,180,377; 6,054,297;5,997,867; and 5,866,692.

The invention also encompasses single chain variable region fragments(“scFv”) of antibodies of this invention, such as mu-anti-OSM-R.beta.Single chain variable region fragments are made by linking light and/orheavy chain variable regions by using a short linking peptide. Bird etal. (1988) Science 242: 423-426 describes example of linking peptideswhich bridge approximately 3.5 nm between the carboxy terminus of onevariable region and the amino terminus of the other variable region.Linkers of other sequences have been designed and used, Bird et al.(1988). Linkers can in turn be modified for additional functions, suchas attachment of drugs or attachment to solid supports. The single chainvariants can be produced either recombinantly or synthetically. Forsynthetic production of scFv, an automated synthesizer can be used. Forrecombinant production of scFv, a suitable plasmid containingpolynucleotide that encodes the scFv can be introduced into a suitablehost cell, either eukaryotic, such as yeast, plant, insect or mammaliancells, or prokaryotic, such as E. coli. Polynucleotides encoding thescFv of interest can be made by routine manipulations such as ligationof polynucleotides. The resultant scFv can be isolated using standardprotein purification techniques known in the art.

The invention includes modifications to OSM-R.beta agonists,antagonists, modulators and antibodies, including functionallyequivalent antibodies and polypeptides that do not significantly affecttheir properties and variants that have enhanced or decreased activity.Modification of polypeptides is routine practice in the art and need notbe described in detail herein. Examples of modified polypeptides includepolypeptides with conservative substitutions of amino acid residues, oneor more deletions or additions of amino acids which do not significantlydeleteriously change the functional activity, or use of chemicalanalogs. Amino acid residues which can be conservatively substituted forone another include but are not limited to: glycine/alanine;valine/isoleucine/leucine; asparagine/glutamine; aspartic acid/glutamicacid; serine/threonine; lysine/arginine; and phenylalanine/tryosine.These polypeptides also include glycosylated and nonglycosylatedpolypeptides, as well as polypeptides with other post-translationalmodifications, such as, for example, glycosylation with differentsugars, acetylation, and phosphorylation. Preferably, the amino acidsubstitutions would be conservative, i.e., the substituted amino acidwould possess similar chemical properties as that of the original aminoacid. Such conservative substitutions are known in the art, and exampleshave been provided above. Amino acid modifications can range fromchanging or modifying one or more amino acids to complete redesign of aregion, such as the variable region. Changes in the variable region canalter binding affinity and/or specificity. Other methods of modificationinclude using coupling techniques known in the art, including, but notlimited to, enzymatic means, oxidative substitution and chelation.Modifications can be used, for example, for attachment of labels forimmunoassay, such as the attachment of radioactive moieties forradioimmunoassay. Modified polypeptides are made using establishedprocedures in the art and can be screened using standard assays known inthe art.

The invention also encompasses fusion proteins comprising one or morefragments or regions from the polypeptides and antibodies of thisinvention. In one embodiment, a fusion polypeptide is provided thatcomprises at least 10 contiguous amino acids of variable light chainregion and at least 10 amino acids of variable heavy chain region. Inanother embodiment, the fusion polypeptide contains a heterologousimmunoglobulin constant region. In another embodiment, the fusionpolypeptide contains a light chain variable region and a heavy chainvariable region of an antibody produced from a hybridoma deposited withthe ATCC as described herein. For purposes of this invention, anantibody fusion protein contains one or more anti-OSM-R.betapolypeptides and another amino acid sequence to which it is not attachedin the native molecule, for example, a heterologous sequence or ahomologous sequence from another region.

An anti-OSM-R.beta polypeptide, and other OSM-R.beta agonists,antagonists and modulators can be created by methods known in the art,for example, synthetically or recombinantly. One method of producingOSM-R.beta peptide agonists, antagonists and modulators involveschemical synthesis of the polypeptide, followed by treatment underoxidizing conditions appropriate to obtain the native conformation, thatis, the correct disulfide bond linkages. This can be accomplished usingmethodologies well known to those skilled in the art (see Kelley, R. F.& Winkler, M. E. in Genetic Engineering Principles and Methods, Setlow,J. K., ed., Plenum Press, N.Y., vol. 12, pp 1-19 (1990); Stewart, J. M.& Young, J. D. Solid Phase Peptide Synthesis Pierce Chemical Co.Rockford, Ill. (1984); see also U.S. Pat. Nos. 4,105,603; 3,972,859;3,842,067; and 3,862,925).

Polypeptides of the invention may be conveniently prepared using solidphase peptide synthesis (Merrifield, J. Am. Chem. Soc., 85:2149 (1964);Houghten, Proc. Natl. Acad. Sci. USA 82:5132 (1985)).

In yet another alternative, fully human antibodies may be obtained byusing commercially available mice that have been engineered to expressspecific human immunoglobulin proteins. Transgenic animals that aredesigned to produce a more desirable (e.g., fully human antibodies) ormore robust immune response may also be used for generation of humanizedor human antibodies. Examples of such technology are Xenomouse™ fromAbgenix, Inc. (Fremont, Calif.) and HuMAb-Mouse® and TC Mouse™ fromMedarex, Inc. (Princeton, N.J.).

In an alternative, antibodies may be made recombinantly and expressedusing any method known in the art. Antibodies may be made recombinantlyby first isolating the antibodies made from host animals, obtaining thegene sequence, and using the gene sequence to express the antibodyrecombinantly in host cells (e.g., CHO cells). Another method that maybe employed is to express the antibody sequence in plants (e.g.,tobacco) or transgenic milk. Methods for expressing antibodiesrecombinantly in plants or milk have been disclosed. See, for example,Peeters, et al. (2001) Vaccine 19:2756; Lonberg, N. and D. Huszar (1995)Int. Rev. Immunol 13:65; and Pollock, et al.(1999) J Immunol Methods231:147. Methods for making derivatives of antibodies, e.g., humanized,single chain, etc. are known in the art. In another alternative,antibodies may be made recombinantly by phage display technology. See,for example, U.S. Pat. Nos. 5,565,332; 5,580,717; 5,733,743; 6,265,150;and Winter et al., Annu. Rev. Immunol. 12:433-455 (1994).

The antibodies or protein of interest may be subjected to sequencing byEdman degradation, which is well known to those of skill in the art. Thepeptide information generated from mass spectrometry or Edmandegradation can be used to design probes or primers that are used toclone the protein of interest.

An alternative method of cloning the protein of interest is by “panning”using purified OSM-R.beta or portions thereof for cells expressing theantibody or protein of interest. The “panning” procedure is conducted byobtaining a cDNA library from tissues or cells that express the antibodyor protein of interest, over-expressing the cDNAs in a second cell type,and screening the transfected cells of the second cell type for aspecific binding to OSM-R.beta. Detailed descriptions of the methodsused in cloning mammalian genes coding for cell surface proteins by“panning” can be found in the art. See, for example, Aruffo, A. andSeed, B. Proc. Natl. Acad. Sci. USA, 84, 8573-8577 (1987) and Stephan,J. et al., Endocrinology 140: 5841-5854 (1999).

cDNAs encoding anti-OSM-R.beta antibodies, and other OSM-R.beta peptideagonists, antagonists and modulators can be obtained by reversetranscribing the mRNAs from a particular cell type according to standardmethods in the art. Specifically, mRNA can be isolated using variouslytic enzymes or chemical solutions according to the procedures setforth in Sambrook, et al. supra or extracted by commercially availablenucleic-acid-binding resins following the accompanying instructionsprovided by manufacturers (e.g., Qiagen, Invitrogen, Promega). Thesynthesized cDNAs are then introduced into an expression vector toproduce the antibody or protein of interest in cells of a second type.It is implied that an expression vector must be replicable in the hostcells either as episomes or as an integral part of the chromosomal DNA.Suitable expression vectors include but are not limited to plasmids,viral vectors, including adenoviruses, adeno-associated viruses,retroviruses, and cosmids.

The vectors containing the polynucleotides of interest can be introducedinto the host cell by any of a number of appropriate means, includingelectroporation, transfection employing calcium chloride, rubidiumchloride, calcium phosphate, DEAE-dextran, or other substances;microprojectile bombardment; lipofection; and infection (e.g., where thevector is an infectious agent such as vaccinia virus). The choice ofintroducing vectors or polynucleotides will often depend on features ofthe host cell.

Any host cells capable of over-expressing heterologous DNAs can be usedfor the purpose of isolating the genes encoding the antibody,polypeptide or protein of interest. Non-limiting examples of mammalianhost cells include but not limited to COS, HeLa, and CHO cells.Preferably, the host cells express the cDNAs at a level of about 5 foldhigher, more preferably 10 fold higher, even more preferably 20 foldhigher than that of the corresponding endogenous antibody or protein ofinterest, if present, in the host cells. Screening the host cells for aspecific binding to OSM-R.beta is effected by an immunoassay or FACS. Acell over-expressing the antibody or protein of interest can beidentified.

Various techniques are also available which may now be employed toproduce mutant OSM-R.beta peptide agonists, antagonists, and modulatorsthat encodes for additions, deletions, or changes in amino acid sequenceof the resultant protein relative to the parent OSM-R.beta peptideagonist, antagonist or modulator molecule.

The invention includes polypeptides comprising an amino acid sequence ofthe antibodies of this invention. The polypeptides of this invention canbe made by procedures known in the art. The polypeptides can be producedby proteolytic or other degradation of the antibodies, by recombinantmethods (i.e., single or fusion polypeptides) as described above or bychemical synthesis. Polypeptides of the antibodies, especially shorterpolypeptides up to about 50 amino acids, are conveniently made bychemical synthesis. Methods of chemical synthesis are known in the artand are commercially available. For example, an anti-OSM-R.betapolypeptide could be produced by an automated polypeptide synthesizeremploying the solid phase method.

IV. Methods for Screening Polypeptides and Monoclonal Antibodies

Several methods may be used to screen polypeptides and monoclonalantibodies that bind to OSM-R.beta. It is understood that “binding”refers to biologically or immunologically relevant binding, i.e.,binding which is specific for the unique antigen for which theimmunoglobulin molecule is encoded, or to which the polypeptide isdirected. It does not refer to non-specific binding that may occur whenan immunoglobulin is used at a very high concentration against anon-specific target. In one embodiment, monoclonal antibodies arescreened for binding to OSM-R.beta using standard screening techniques.In this manner, anti-OSM-R.beta monoclonal antibody was obtained. Inaccordance with the Budapest Treaty, a hybridoma which producesanti-OSM-R.beta monoclonal antibodies has been deposited in the AmericanType Culture Collection (ATCC) 10801 University Blvd., Manassas Va.20110-2209 on Jan. 12, 2005 with a Patent Deposit Designation ofPTA-6511.

Monoclonal antibodies that bind to OSM-R.beta are screened for bindingto cancerous tissues and non-cancerous cells. In one embodiment,monoclonal antibodies which bind to OSM-R.beta and that are also crossreactive to human cancerous cells or tissues, but not to normal cells ortissues to the same degree, are selected. One method that may beemployed for screening is immunohistochemistry (IHC). Standardimmunohistochemical techniques are known to those of average skill inthe art. See, for example, Animal Cell Culture Methods (J.P. Mather andD. Barnes, eds., Academic Press, Vol. 57, Ch. 18 and 19, pp. 314-350,1998). Biological samples (e.g., tissues) may be obtained from biopsies,autopsies, or necropsies. To ascertain if OSM-R.beta is present only oncancerous cells, anti-OSM-R.beta antibodies may be used to detect thepresence of OSM-R.beta on tissues from individuals with cancer whileother non-cancerous tissues from the individual suffering from cancer ortissues from individuals without cancer are used as a control. Thetissue can be embedded in a solid or semi-solid substance that preventsdamage during freezing (e.g., agarose gel or OCT) and then sectioned forstaining. Cancers from different organs and at different grades can beused to screen monoclonal antibodies. Examples of tissues that may beused for screening purposes include but are not limited to ovary,breast, lung, prostate, colon, kidney, skin, thyroid, brain, heart,liver, stomach, nerve, blood vessels, bone, upper digestive tract, andpancreas. Examples of different cancer types that may be used forscreening purposes include but are not limited to carcinomas,adenocarcinomas, sarcomas, adenosarcomas, lymphomas, and leukemias.

In yet another alternative, cancerous cells lines such as SK-Ov-3 (ATCC#HTB 77), LnCap (ATCC #CRL-1740), A549 (ATCC #CCL 185), PANC-1 (ATCC#CRL 1469), SK-BR-3 (ATCC #HTB 30), SK-MES-1 (ATCC #HTB 58), HT-29(HTB-38), SW 480 (ATCC #CCL 228), AsPC-1 (ATCC #CRL 1682), Capan-1 (ATCC#HTB 79), CFPAC-1 (ATCC #CRL 1918), HPAF-II (ATCC #CRL-1997), Hs-700T(ATCC #HTB 147), ES-2 (ATCC #CRL-1978), PC-3 (ATCC #CRL 1435), Du-145(ATCC #HTB-81), CaLu3 (ATCC #HTB-55), A498 (ATCC # CRL-7908), Caki-2(ATCC # HTB-47), 786-O (ATCC # CRL-1932), Hs 766T (ATCC # HTB-134),MCF7(ATCC # HTB-22), BT-474 (ATCC # HTB-20), Rav CA130 (proprietary lungcancer line developed at Raven Biotechnologies, inc.), Rav9926(proprietary pancreatic cancer cell line developed at Raven), and22Rv1(ATCC #CRL-2505) and normal cells from their respective tissues maybe used to screen for monoclonal antibodies which are specific forcancerous tissue. Primary, or low passage, cell cultures derived fromnormal tissues from different organs, including but not limited to,ovary, breast, lung, prostate, colon, kidney, skin, thyroid, aorticsmooth muscle, and endothelial cells can be used as negative controls.The cancerous or non-cancerous cells can be grown on glass slides orcoverslips, or on plastic surfaces, or prepared in a CellArray™, asdescribed in WO 01/43869, and screened for the binding of antibody usingIHC as described above for tissues. Alternatively, cells may be removedfrom the growth surface using non-proteolytic means and spun into apellet, which is then embedded and treated as tissues for IHC analysisas described above. Cells may be inoculated into immunodeficientanimals, a tumor allowed to grow, and then this tumor may be harvested,embedded, and used as a tissue source for IHC analysis. In anotheralternative, single cells may be screened by incubating with the primaryantibody, a secondary “reporter” antibody linked to a fluorescentmolecule and then analyzed using a fluorescent activated cell-sorting(FACS) machine.

In yet another alternative, live cell ELISAs can be used to screen forantibodies that are specific for cancer cell lines (such as the oneslisted above) and normal cells from their respective tissues. Cells maybe plated onto 96-well plates and then grown to confluency. The growthmedium is then removed and the cells are blocked with blocking buffer.Primary antibodies may be added and then the plates washed and secondaryantibodies added and the plates may be developed by adding a substrateand color change can be detected using a plate reader.

Several different detection systems may be utilized to detect binding ofantibodies to tissue section. Typically, immunohistochemistry involvesthe binding of a primary antibody to the tissue and then a secondaryantibody reactive against the species from the primary antibody wasgenerated and conjugated to a detectable marker (e.g., horseradishperoxidase, HRP, or diaminobenzedine, DAB). One alternative method thatmay be used is polyclonal mirror image complementary antibodies orpolyMICA. PolyMICA (polyclonal Mirror Image Complementary Antibodies)technique, described by D. C. Mangham and P. G. Isaacson (Histopathology(1999) 35(2):129-33), can be used to test binding of primary antibodies(e.g., anti-OSM-R.beta antibodies) to normal and cancerous tissue.Several kinds of polyMICA™ Detection kits are commercially availablefrom The Binding Site Limited (P.O. Box 4073 Birmingham B29 6ATEngland). Product No. HK004.D is a polyMICA™ Detection kit which usesDAB chromagen. Product No. HK004.A is a polyMICA™ Detection kit whichuses AEC chromagen. Alternatively, the primary antibody may be directlylabeled with the detectable marker.

The first step in IHC screening to select for an appropriate antibody isthe binding of primary antibodies raised in mice (e.g., anti-OSM-R.betaantibodies) to one or more immunogens (e.g., cells or tissue samples).In one embodiment, the tissue sample is sections of frozen tissue fromdifferent organs. The cells or tissue samples can be either cancerous ornon-cancerous.

Frozen tissues can be prepared, sectioned, with or without fixation, andIHC performed by any of a number of methods known to one familiar withthe art. See, for example, Stephan et al. Dev. Biol. 212: 264-277(1999), and Stephan et al. Endocrinology 140: 5841-54 (1999).

V. Methods of Characterizing Anti-OSM-R.beta Antibodies

Several methods can be used to characterize anti-OSM-R.beta antibodies.One method is to identify the epitope to which it binds. Epitope mappingis commercially available from various sources, for example, PepscanSystems (Edelhertweg 15, 8219 PH Lelystad, The Netherlands). Epitopemapping can be used to determine the sequence to which ananti-OSM-R.beta antibody binds. The epitope can be a linear epitope,i.e., contained in a single stretch of amino acids, or a conformationalepitope formed by a three-dimensional interaction of amino acids thatmay not necessarily be contained in a single stretch. Peptides ofvarying lengths (e.g., at least 4-6 amino acids long) can be isolated orsynthesized (e.g., recombinantly) and used for binding assays withanti-OSM-R.beta antibody. The epitope to which anti-OSM-R.beta antibodybinds can be determined in a systematic screening by using overlappingpeptides derived from the extracellular sequence and determining bindingby anti-OSM-R.beta antibody.

Yet another method that can be used to characterize an anti-OSM-R.betaantibody is to use competition assays with other antibodies known tobind to the same antigen, i.e., OSM-R.beta to determine ifanti-OSM-R.beta antibodies binds to the same epitope as otherantibodies. Examples of commercially available antibodies to OSM-R.betamay be available and may be identified using the binding assays taughtherein. Competition assays are well known to those of skill in the art,and such procedures and illustrative data are detailed further in theExamples. Anti-OSM-R.beta antibodies can be further characterized by thetissues, type of cancer or type of tumor to which they bind.

Another method of characterizing anti-OSM-R.beta antibodies is by theantigen to which it binds. Anti-OSM-R.beta antibodies were used inWestern blots with cell lysates from various human cancers. As is knownto one of skill in the art, Western blotting can involve running celllysates and/or cell fractions on a denaturing or non-denaturing gel,transferring the proteins to nitrocellulose paper, and then probing theblot with an antibody (e.g., anti-OSM-R.beta antibody) to see whichproteins are bound by the antibody. This procedure is detailed furtherin the Examples.

VI. Methods of Diagnosing Cancer Using Anti-OSM-R.beta Antibodies andOSM-R.beta Modulators

Monoclonal antibodies to OSM-R.beta made by the methods disclosed hereinmay be used to identify the presence or absence of cancerous cells in avariety of tissues, including but not limited to, ovary, breast, lung,prostate, colon, kidney, pancreas, skin, thyroid, brain, heart, liver,stomach, nerve, blood vessels, bone, and upper digestive tract, forpurposes of diagnosis. Monoclonal antibodies to OSM-R.beta made by themethods disclosed herein may also be used to identify the presence orabsence of cancerous cells, or the level thereof, which are circulatingin blood after their release from a solid tumor. Such circulatingantigen may be an intact OSM-R.beta antigen, or a fragment thereof thatretains the ability to be detected according to the methods taughtherein. Such detection may be effected by FACS analysis using standardmethods commonly used in the art.

These uses can involve the formation of a complex between OSM-R.beta andan antibody that binds specifically to OSM-R.beta. Examples of suchantibodies include but are not limited to those anti-OSM-R.betamonoclonal antibodies produced by the hybridoma deposited in the ATCCwith the designation PTA-6511. The formation of such a complex can be invitro or in vivo. Without being bound by theory, monoclonal antibodyanti-OSM-R.beta can bind to OSM-R.beta through the extracellular domainof OSM-R.beta and may then be internalized.

In a preferred embodiment of the diagnostic methods of this invention,the antibody bears a detectable label. Examples of labels that may beused include a radioactive agent or a fluorophore, such asfluoroisothiocyanate or phycoerythrin.

As with other known antibodies used commercially for diagnostic andtherapeutic purposes, the target antigen of this invention is broadlyexpressed in normal tissue. It is also up regulated in some tumors.Therefore, the particular dosages and routes of delivery of theantibodies of this invention as used for diagnostic or therapeuticagents will be tailored to the particular tumor or disease state athand, as well as to the particular individual being treated.

One method of using the antibodies for diagnosis is in vivo tumorimaging by linking the antibody to a radioactive or radioopaque agent,administering the antibody to the individual and using an x-ray or otherimaging machine to visualize the localization of the labeled antibody atthe surface of cancer cells expressing the antigen. The antibody isadministered at a concentration that promotes binding at physiologicalconditions.

In vitro techniques for detection of OSM-R.beta are routine in the artand include enzyme linked immunosorbent assays (ELISAs),immunoprecipitations, immunofluorescence, enzyme immunoassay (EIA),radioimmunoassay (RIA), and Western blot analysis.

In aspects of this invention, methods of radioimaging of tumours orneoplasms, or of measuring the effectiveness of a method of treatmentwith a radiolabelled antibody, comprising the step of administering aradiolabelled, tumour-specific antibody to an individual following thepractice of this invention. The radiolabelled antibody may be amonoclonal or polyclonal antibody comprising a radiolabel, preferablyselected from the group consisting of Technetium-99m, Indium-111,Iodine-131, Rhenium-186, Rhenium-188, Samarium-153, Lutetium-177,Copper-64, Scandium-47, Yttrium-90. Monoclonal antibodies labelled withtherapeutic radionuclides such as Iodine-131, Rhenium-188, Holmium-166,Samarium-153 and Scandium-47, which do not compromise theimmunoreactivity of antibodies and are not broken down in vivo, areespecially preferred. The person skilled in the art will appreciate thatother radioactive isotopes are known, and may be suitable for specificapplications. The radioimaging may be conducted using Single PhotonEmission Computer Tomography (SPECT), Position Emmission Tomography(PET), Computer Tomography (CT) or Magnetic Resonance Imaging (MRI).Correlative imaging, which permits greater anatomical definition oflocation of metastases located by radioimmunoimaging, is alsocontemplated.

In other methods, the cancerous cells are removed and the tissueprepared for immunohistochemistry by methods well known in the art(e.g., embedding in a freezing compound, freezing and sectioning, withor without fixation; fixation and paraffin embedding with or withoutvarious methods of antigen retrieval and counterstaining). Themonoclonal antibodies may also be used to identify cancerous cells atdifferent stages of development. The antibodies may also be used todetermine which individuals' tumors express the antigen on their surfaceat a pre-determined level and are thus candidates for immunotherapyusing antibodies directed against said antigen. The antibodies mayrecognize both primary and metastasizing cancers of the ovary, prostateand pancreas and primary cancers of the lung that express OSM-R.beta. Asused herein, detection may include qualitative and/or quantitativedetection and may include comparing the level measured to a normal cellfor an increased level of expression of OSM-R.beta in cancerous cells.

The invention also provides methods of aiding diagnosis of cancer (suchas ovarian, lung, pancreatic, prostate, colon, or breast cancer) in anindividual using any antibody that binds to OSM-R.beta and any othermethods that can be used determine the level of OSM-R.beta expression.As used herein, methods for “aiding diagnosis” means that these methodsassist in making a clinical determination regarding the classification,or nature, of cancer, and may or may not be conclusive with respect tothe definitive diagnosis. Accordingly, a method of aiding diagnosis ofcancer can comprise the step of detecting the level of OSM-R.beta in abiological sample from the individual and/or determining the level ofOSM-R.beta expression in the sample. Antibodies recognizing the antigenor a portion thereof may also be used to create diagnostic immunoassaysfor detecting antigen released or secreted from living or dying cancercells in bodily fluids, including but not limited to, blood, saliva,urine, pulmonary fluid, or ascites fluid.

Not all cells in a particular tumor of interest will express OSM-R.beta,and cancerous cells in other tissues may express OSM-R.beta, thus anindividual should be screened for the presence or absence of OSM-R.betaon cancerous cells to determine the usefulness of immunotherapy in theindividual. The anti-OSM-R.beta antibodies made by the methods disclosedherein may be used to determine whether an individual diagnosed withcancer may be deemed a candidate for immunotherapy using antibodiesdirected against OSM-R.beta. In one embodiment, a cancerous tumor or abiopsy sample may be tested for expression of OSM-R.beta, usingantibodies directed against OSM-R.beta. Individuals with cancer cellsthat express OSM-R.beta are suitable candidates for immunotherapy usingantibodies directed against OSM-R.beta. Staining with anti-OSM-R.betaantibody may also be used to distinguish cancerous tissues from normaltissues.

The therapeutic agents of this invention are particularly preferred forthe diagnosis and treatment of various human cancers of differenttissues including but not limited to lung carcinomas, melanomas, breastcarcinomas, ovarian carcinomas, neuroblastomas and glioblastomas, andless associated with colon or prostate carcinomas. They are also usefulin the regulation of immune function, thrombopoiesis, cholesterolhomeostasis and neurotrophic mediators.

Methods of using anti-OSM-R.beta antibodies for diagnostic purposes areuseful both before and after any form of anti-cancer treatment, e.g.,chemotherapy or radiation therapy, to determine which tumors are mostlikely to respond to a given treatment, prognosis for individual withcancer, tumor subtype or origin of metastatic disease, and progressionof the disease or response to treatment.

The compositions of this invention are also suitable for diagnosis ofdisease states other than cancer, using the methods generally describedabove in application with other diseased (non-cancerous) cells. Diseasestates suitable for use in the methods of this invention include, butare not limited to, diseases or disorders associated with inflammatoryor autoimmune responses in individuals. The methods described above maybe used for modulating inflammatory or autoimmune responses inindividuals. Diseases and conditions resulting from inflammation andautoimmune disorders that may be subject to diagnosis and/or treatmentusing the compositions and methods of the invention include, by way ofillustration and not of limitation, multiple sclerosis, meningitis,encephalitis, stroke, other cerebral traumas, inflammatory bowel diseaseincluding ulcerative colitis and Crohn's disease, myasthenia gravis,lupus, rheumatoid arthritis, asthma, acute juvenile onset diabetes, AIDSdementia, atherosclerosis, nephritis, retinitis, atopic dermatitis,psoriasis, myocardial ischemia and acute leukocyte-mediated lung injury.

Still other indications for diagnostic and/or therapeutic use ofantibodies and other therapeutic agents of the invention includeadministration to individuals at risk of organ or graft rejection. Overrecent years there has been a considerable improvement in the efficiencyof surgical techniques for transplanting tissues and organs such asskin, kidney, liver, heart, lung, pancreas and bone marrow. Perhaps theprincipal outstanding problem is the lack of satisfactory agents forinducing immunotolerance in the recipient to the transplanted allograftor organ. When allogeneic cells or organs are transplanted into a host(i.e., the donor and donee are different individuals from the samespecies), the host immune system is likely to mount an immune responseto foreign antigens in the transplant (host-versus-graft disease)leading to destruction of the transplanted tissue.

Uses described anywhere in this application that recite their use foranti-OSM-R.beta antibodies also encompass the use of other OSM-R.betaagonists, antagonists and modulators as described herein. In suchembodiments, the OSM-R.beta agonists, antagonist or other non-antibodymodulator is substituted for the OSM-R.beta antibody in the stepsdescribed, and alterations within the scope of the ordinarily skilledpractitioner are made to tailor the method to the substituted OSM-R.betamodulatory composition.

VII. Compositions of this Invention

This invention also encompasses compositions, including pharmaceuticalcompositions, comprising anti-OSM-R.beta antibodies, polypeptidesderived from anti-OSM-R.beta antibodies, polynucleotides comprisingsequence encoding anti-OSM-R.beta antibodies, and other agents asdescribed herein. As used herein, compositions further comprises one ormore antibodies, polypeptides and/or proteins that bind to OSM-R.beta,OSM-R.beta agonists, antagonists, modulators, and/or one or morepolynucleotides comprising sequences encoding one or more antibodies,polypeptides and proteins that bind to OSM-R.beta.

The invention further provides for conjugates of any OSM-R.beta peptideagonist, antagonist or modulator, and additional chemical structuresthat support the intended function or functions of the particularOSM-R.beta peptide agonist, antagonist or modulator. These conjugatesinclude OSM-R.beta peptide agonist, antagonist or modulator covalentlybound to a macromolecule such as any insoluble, solid support matrixused in the diagnostic, screening or purification procedures discussedherein. Suitable matrix materials include any substance that ischemically inert, has high porosity and has large numbers of functionalgroups capable of forming covalent linkages with peptide ligands.Examples of matrix materials and procedures for preparation ofmatrix-ligand conjugates are described in Dean et al. (eds) AffinityChromatography: A Practical Approach, IRL Press (1985); Lowe, “AnIntroduction to Affinity Chromatography”, in Work et al. (eds)Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 7,Part II, North-Holland (1979); Porath et al., “Biospecific AffinityChromatography”, in Neurath et al. (eds), The Proteins, 3rd ed., Vol. 1,pp. 95-178 (1975); and Schott, Affinity Chromatography, Dekker (1984).

Also provided herein are conjugates of OSM-R.beta peptide agonist,antagonist or modulator and any reporter moiety used in the diagnosticprocedures discussed herein.

The OSM-R.beta peptide agonist, antagonist or modulator agents,polypeptides and proteins of this invention, including anti-OSM-R.betaantibodies, are further identified and characterized by any (one ormore) of the following criteria: (a) ability to bind to OSM-R.beta(including OSM-R.beta on cancer cells, including but not limited toovarian, prostate, pancreatic, lung, colon, or breast cancer cells); (b)ability to competitively inhibits preferential binding of a knownanti-OSM-R.beta antibody to OSM-R.beta, including the ability topreferentially bind to the same OSM-R.beta epitope to which the originalantibody preferentially binds; (c) ability to bind to a portion ofOSM-R.beta that is exposed on the surface of a living cell in vitro orin vivo; (d) ability to bind to a portion of OSM-R.beta that is exposedon the surface of living cancer cells, such as but not limited toovarian, prostate, pancreatic, lung, colon, or breast cancer cells; (e)ability to deliver a chemotherapeutic agent or detectable marker tocancerous cells (such as but not limited to ovarian, prostate,pancreatic, lung, colon, or breast cancer cells) expressing OSM-R.beta;(f) ability to deliver a therapeutic agent into cancerous cells (such asbut not limited to ovarian cancer cells) expressing OSM-R.beta.

In some embodiments, the antibody of the invention is an antibody thatis produced by a host cell with a deposit number of ATCC No. PTA-6511,or progeny thereof. The present invention also encompasses variousformulations of antibodies produced by these deposited hybridomas andequivalent antibodies or polypeptide fragments (e.g., Fab, Fab′,F(ab′)₂, Fv, Fc, etc.), chimeric antibodies, single chain (ScFv),mutants thereof, fusion proteins comprising an antibody portion,humanized antibodies, and any other modified configuration of any ofthese or equivalent antibodies that comprises an antigen (OSM-R.beta),recognition site of the required specificity. The invention alsoprovides human antibodies displaying one or more of the biologicalcharacteristics of an anti-OSM-R.beta family member. The equivalentantibodies of the anti-OSM-R.beta family (including humanized antibodiesand human antibodies), polypeptide fragments, and polypeptidescomprising any of these fragments are identified and characterized byany (one or more) of the five criteria described above.

In some embodiments, the antibodies, polypeptides and proteins of theinvention that bind to OSM-R.beta are antibodies, polypeptides andproteins that competitively inhibit preferential binding of aherein-specified anti-OSM-R.beta antibody to OSM-R.beta. In someembodiments, the antibodies, the polypeptides and the proteinspreferentially bind to the same epitope on OSM-R.beta as the antibodymu-anti-OSM-R.beta preferentially binds.

Accordingly, the invention provides any of the following (orcompositions, including pharmaceutical compositions, comprising any ofthe following): (a) an antibody produced by the host cell with a depositnumber identified above or its progeny; (b) a humanized form of such anantibody; (c) an antibody comprising one or more of the light chainand/or heavy chain variable regions of such an antibody; (d) a chimericantibody comprising variable regions homologous or derived from variableregions of a heavy chain and a light chain of such an antibody, andconstant regions homologous or derived from constant regions of a heavychain and a light chain of a human antibody; (e) an antibody comprisingone or more of the light chain and/or heavy chain CDRs (at least one,two, three, four, five, or six) of such an antibody; (f) an antibodycomprising a heavy and/or a light chain of such an antibody; (g) a humanantibody that is equivalent to such an antibody. A humanized form of theantibody may or may not have CDRs identical to that original antibody,or antibody produced by a host cell with a deposit number identifiedabove. Determination of CDR regions is well within the skill of the art.In some embodiments, the invention provides an antibody which comprisesat least one CDR that is substantially homologous to at least one CDR,at least two, at least three, at least four, at least 5 CDRs of anantibody produced by one of the above-identified deposited hybridomas(or, in some embodiments substantially homologous to all 6 CDRs of oneof these antibodies, or derived from one of these antibodies), orantibody produced by the host cell with a deposit number identifiedabove. Other embodiments include antibodies that have at least two,three, four, five, or six CDR(s) that are substantially homologous to atleast two, three, four, five or six CDRs of an antibody produced from ahybridoma deposited as identified herein, or derived from such anantibody. It is understood that, for purposes of this invention, bindingspecificity and/or overall activity (which may be in terms of deliveringa chemotherapeutic agent to or into cancerous cells to reduce the growthand/or proliferation of cancer cells, to induce apoptotic cell death inthe cancer cell, to delay the development of metastasis, and/or treatingpalliatively) is generally retained, although the extent of activity mayvary compared to an antibody produced by a deposited hybridoma (may begreater or lesser). The invention also provides methods of making any ofthese antibodies. Methods of making antibodies are known in the art andare described herein.

The invention also provides polypeptides comprising an amino acidsequence of the antibodies of the invention. In some embodiments, thepolypeptide comprises one or more of the light chain and/or heavy chainvariable regions of the antibody. In some embodiments, the polypeptidecomprises one or more of the light chain and/or heavy chain CDRs of theantibody. In some embodiments, the polypeptide comprises three CDRs ofthe light chain and/or heavy chain of the antibody. In some embodiments,the polypeptide comprises an amino acid sequence of the antibody thathas any of the following: at least 5 contiguous amino acids of asequence of the original antibody, at least 8 contiguous amino acids, atleast about 10 contiguous amino acids, at least about 15 contiguousamino acids, at least about 20 contiguous amino acids, at least about 25contiguous amino acids, at least about 30 contiguous amino acids,wherein at least 3 of the amino acids are from a variable region of theantibody. In one embodiment, the variable region is from a light chainof the original antibody. In another embodiment, the variable region isfrom a heavy chain of the antibody. In another embodiment, the 5 (ormore) contiguous amino acids are from a complementarity-determiningregion (CDR) of the antibody.

In some embodiments of this invention, cells of this invention thatexpress OSM-R.beta, a portion of OSM-R.beta, anti-OSM-R.beta antibodiesor other OSM-R.beta-binding polypeptides of this invention areadministered directly to an individual to modulate that individual'sendogenous in vivo OSM-R.beta biological activity.

VIII. Methods of Using OSM-R.beta Modulators and Anti-OSM-R.betaAntibodies for Therapeutic Purposes

Monoclonal antibodies to OSM-R.beta may be used for therapeutic purposesin individuals with cancer or other diseases. Therapy withanti-OSM-R.beta antibodies can involve formation of complexes both invitro and in vivo as described above. In one embodiment, monoclonalantibody anti-OSM-R.beta can bind to and reduce the proliferation ofcancerous cells. It is understood that the antibody is administered at aconcentration that promotes binding at physiological (e.g., in vivo)conditions. In another embodiment, monoclonal antibodies to OSM-R.betacan be used for immunotherapy directed at cancerous cells of differenttissues such as colon, lung, breast, prostate, ovary, pancreas, kidneyand other types of cancer such as sarcoma. In another embodiment,monoclonal antibody anti-OSM-R.beta alone can bind to and reduce celldivision in the cancer cell. In another embodiment, monoclonal antibodyanti-OSM-R.beta can bind to cancerous cells and prevent or delay thedevelopment of metastasis. In yet another embodiment, an individual withcancer is given palliative treatment with anti-OSM-R.beta antibody.Palliative treatment of a cancer individual involves treating orlessening the adverse symptoms of the disease, or iatrogenic symptomsresulting from other treatments given for the disease without directlyaffecting the cancer progression. This includes treatments for easing ofpain, nutritional support, sexual problems, psychological distress,depression, fatigue, psychiatric disorders, nausea, vomiting, etc.

In such situations, the anti-OSM-R.beta antibody may be administeredwith agents that enhance or direct an individual's own immune response,such as an agent that strengthens antibody-dependent cellularcytotoxicity (ADCC).

In other embodiments, at least one fucose residue present in ananti-OSM-R.beta antibody is removed from the oligosaccharides of thatantibody, a modification to enhance ADCC. In similar embodiments, fucoseresidues present in an anti-OSM-R.beta antibody are modified to altertheir composition to the extent required to enhance ADCC compared to theoriginal, unmodified antibody.

In yet another embodiment, anti-OSM-R.beta antibody be conjugated to orassociated with a radioactive molecule, toxin (e.g., calicheamicin),chemotherapeutic molecule, liposomes or other vesicles containingchemotherapeutic compounds and administered to an individual in need ofsuch treatment to target these compounds to the cancer cell containingthe antigen recognized by the antibody and thus eliminate cancerous ordiseased cells. Without being limited to any particular theory, theanti-OSM-R.beta antibody is internalized by the cell bearing OSM-R.betaat its surface, thus delivering the conjugated moiety to the cell toinduce the therapeutic effect. In yet another embodiment, the antibodycan be employed as adjuvant therapy at the time of the surgical removalof a cancer expressing the antigen in order to delay the development ofmetastasis. The antibody can also be administered before surgery(neoadjuvant therapy) in an individual with a tumor expressing theantigen in order to decrease the size of the tumor and thus enable orsimplify surgery, spare tissue during surgery, and/or decrease theresulting disfigurement.

Cell cycle dosing is contemplated in the practice of this invention. Insuch embodiments, a chemotherapeutic agent is used to synchronize thecell cycle of the tumor or other target diseased cells at apre-determined stage. Subsequently, administration of theanti-OSM-R.beta antibody of this invention (alone or with an additionaltherapeutic moiety) is made. In alternative embodiments, ananti-OSM-R.beta antibody is used to synchronize the cell cycle andreduce cell division prior to administration of a second round oftreatment; the second round may be administration of an anti-OSM-R.betaantibody and/or an additional therapeutic moiety.

Chemotherapeutic agents include radioactive molecules, toxins, alsoreferred to as cytotoxins or cytotoxic agents, which includes any agentthat is detrimental to the viability of cancerous cells, agents, andliposomes or other vesicles containing chemotherapeutic compounds.Examples of suitable chemotherapeutic agents include but are not limitedto 1-dehydrotestosterone, 5-fluorouracil decarbazine, 6-mercaptopurine,6-thioguanine, actinomycin D, adriamycin, aldesleukin, alkylatingagents, allopurinol sodium, altretamine, amifostine, anastrozole,anthramycin (AMC)), anti-mitotic agents, cis-dichlorodiamine platinum(II) (DDP) cisplatin), diamino dichloro platinum, anthracyclines,antibiotics, antimetabolites, asparaginase, BCG live (intravesical),betamethasone sodium phosphate and betamethasone acetate, bicalutamide,bleomycin sulfate, busulfan, calcium leucouorin, calicheamicin,capecitabine, carboplatin, lomustine (CCNU), carmustine (BSNU),Chlorambucil, Cisplatin, Cladribine, Colchicin, conjugated estrogens,Cyclophosphamide, Cyclothosphamide, Cytarabine, Cytarabine, cytochalasinB, Cytoxan, Dacarbazine, Dactinomycin, dactinomycin (formerlyactinomycin), daunirubicin HCL, daunorucbicin citrate, denileukindiftitox, Dexrazoxane, Dibromomannitol, dihydroxy anthracin dione,Docetaxel, dolasetron mesylate, doxorubicin HCL, dronabinol, E. coliL-asparaginase, emetine, epoetin alfa, Erwinia L-asparaginase,esterified estrogens, estradiol, estramustine phosphate sodium, ethidiumbromide, ethinyl estradiol, etidronate, etoposide citrororum factor,etoposide phosphate, filgrastim, floxuridine, fluconazole, fludarabinephosphate, fluorouracil, flutamide, folinic acid, gemcitabine HCL,glucocorticoids, goserelin acetate, gramicidin D, granisetron HCL,hydroxyurea, idarubicin HCL, ifosfamide, interferon alfa-2b, irinotecanHCL, letrozole, leucovorin calcium, leuprolide acetate, levamisole HCL,lidocaine, lomustine, maytansinoid, mechlorethamine HCL,medroxyprogesterone acetate, megestrol acetate, melphalan HCL,mercaptipurine, mesna, methotrexate, methyltestosterone, mithramycin,mitomycin C, mitotane, mitoxantrone, nilutamide, octreotide acetate,ondansetron HCL, paclitaxel, pamidronate disodium, pentostatin,pilocarpine HCL, plimycin, polifeprosan 20 with carmustine implant,porfimer sodium, procaine, procarbazine HCL, propranolol, rituximab,sargramostim, streptozotocin, tamoxifen, taxol, teniposide, tenoposide,testolactone, tetracaine, thioepa chlorambucil, thioguanine, thiotepa,topotecan HCL, toremifene citrate, trastuzumab, tretinoin, valrubicin,vinblastine sulfate, vincristine sulfate, and vinorelbine tartrate.

In a preferred embodiment, the cytotoxin is especially effective individing or rapidly dividing cells, such that non-dividing cells arerelatively spared from the toxic effects.

The antibodies of the invention can be internalized within the diseasedor carcinoma cells to which they bind and are therefore particularlyuseful for therapeutic applications, for example, delivering into thecells toxins that need to be internalized for their adverse activity.Examples of such toxins include, but not limited to, saporin,calicheamicin, auristatin, and maytansinoid.

The antibodies or polypeptides of the invention can be associated(including conjugated or linked) to a radioactive molecule, a toxin, orother therapeutic agents, or to liposomes or other vesicles containingtherapeutic agents covalently or non-covalently, directly or indirectly.The antibody may be linked to the radioactive molecule, the toxin, orthe chemotherapeutic molecule at any location along the antibody so longas the antibody is able to bind its target OSM-R.beta.

A toxin or a chemotherapeutic agent may be coupled (e.g., covalentlybonded) to a suitable monoclonal antibody either directly or indirectly(e.g., via a linker group, or, alternatively, via a linking moleculewith appropriate attachment sites, such as a platform molecule asdescribed in U.S. Pat. No. 5,552,391). The toxin and chemotherapeuticagent of the present invention can be coupled directly to the particulartargeting proteins using methods known in the art. For example, a directreaction between an agent and an antibody is possible when eachpossesses a substituent capable of reacting with the other. For example,a nucleophilic group, such as an amino or sulfhydryl group, on one maybe capable of reacting with a carbonyl-containing group, such as ananhydride or an acid halide, or with an alkyl group containing a goodleaving group (e.g., a halide) on the other.

The antibodies or polypeptides can also be linked to a chemotherapeuticagent via a microcarrier. Microcarrier refers to a biodegradable or anon-biodegradable particle which is insoluble in water and which has asize of less than about 150, 120 or 100 mm in size, more commonly lessthan about 50-60 μm, preferably less than about 10, 5, 2.5, 2 or 1.5 μm.Microcarriers include “nanocarriers”, which are microcarriers have asize of less than about 1 μm, preferably less than about 500 nm. Suchparticles are known in the art. Solid phase microcarriers may beparticles formed from biocompatible naturally occurring polymers,synthetic polymers or synthetic copolymers, which may include or excludemicrocarriers formed from agarose or cross-linked agarose, as well asother biodegradable materials known in the art. Biodegradable solidphase microcarriers may be formed from polymers which are degradable(e.g., poly(lactic acid), poly(glycolic acid) and copolymers thereof) orerodible (e.g., poly(ortho esters such as3,9-diethylidene-2,4,8,10-tetraoxaspiro[5.5]undecane (DETOSU) orpoly(anhydrides), such as poly(anhydrides) of sebacic acid) undermammalian physiological conditions. Microcarriers may also be liquidphase (e.g., oil or lipid based), such liposomes, iscoms(immune-stimulating complexes, which are stable complexes ofcholesterol, and phospholipid, adjuvant-active saponin) without antigen,or droplets or micelles found in oil-in-water or water-in-oil emulsions,provided the liquid phase microcarriers are biodegradable. Biodegradableliquid phase microcarriers typically incorporate a biodegradable oil, anumber of which are known in the art, including squalene and vegetableoils. Microcarriers are typically spherical in shape, but microcarriersthat deviate from spherical shape are also acceptable (e.g., elipsoid,rod-shaped, etc.). Due to their insoluble nature (with respect towater), microcarriers are filterable from water and water-based(aqueous) solutions.

The antibody or polypeptide conjugates of the present invention mayinclude a bifunctional linker that contains both a group capable ofcoupling to a toxic agent or chemotherapeutic agent and a group capableof coupling to the antibody. A linker can function as a spacer todistance an antibody from an agent in order to avoid interference withbinding capabilities. A linker can be cleavable or non-cleavable. Alinker can also serve to increase the chemical reactivity of asubstituent on an agent or an antibody, and thus increase the couplingefficiency. An increase in chemical reactivity may also facilitate theuse of agents, or functional groups on agents, which otherwise would notbe possible. The bifunctional linker can be coupled to the antibody bymeans that are known in the art. For example, a linker containing anactive ester moiety, such as an N-hydroxysuccinimide ester, can be usedfor coupling to lysine residues in the antibody via an amide linkage. Inanother example, a linker containing a nucleophilic amine or hydrazineresidue can be coupled to aldehyde groups produced by glycolyticoxidation of antibody carbohydrate residues. In addition to these directmethods of coupling, the linker can be indirectly coupled to theantibody by means of an intermediate carrier such as an aminodextran. Inthese embodiments the modified linkage is via either lysine,carbohydrate, or an intermediate carrier. In one embodiment, the linkeris coupled site-selectively to free thiol residues in the protein.Moieties that are suitable for selective coupling to thiol groups onproteins are well known in the art. Examples include disulfidecompounds, α-halocarbonyl and α-halocarboxyl compounds, and maleimides.When a nucleophilic amine function is present in the same molecule as anα-halo carbonyl or carboxyl group the potential exists for cyclizationto occur via intramolecular alkylation of the amine. Methods to preventthis problem are well known to one of ordinary skill in the art, forexample by preparation of molecules in which the amine and α-halofunctions are separated by inflexible groups, such as aryl groups ortrans-alkenes, that make the undesired cyclization stereochemicallydisfavored. See, for example, U.S. Pat. No. 6,441,163 for preparation ofconjugates of maytansinoids and antibody via a disulfide moiety.

One of the cleavable linkers that can be used for the preparation ofantibody-drug conjugates is an acid-labile linker based on cis-aconiticacid that takes advantage of the acidic environment of differentintracellular compartments such as the endosomes encountered duringreceptor mediated endocytosis and the lysosomes. See, for example, Shenet al., Biochem. Biophys. Res. Commun. 102:1048-1054 (1981) for thepreparation of conjugates of daunorubicin with macromolecular carriers;Yang et al., J. Natl. Canc. Inst. 80:1154-1159 (1988) for thepreparation of conjugates of daunorubicin to an anti-melanoma antibody;Dillman et al., Cancer Res. 48:6097-6102 (1988) for using an acid-labilelinker in a similar fashion to prepare conjugates of daunorubicin withan anti-T cell antibody; Trouet et al., Proc. Natl. Acad. Sci.79:626-629 (1982) for linking daunorubicin to an antibody via a peptidespacer arm.

An antibody (or polypeptide) of this invention may be conjugated(linked) to a radioactive molecule by any method known to the art. For adiscussion of methods for radiolabeling antibody see “Cancer Therapywith Monoclonal AntibodiesT”, D. M. Goldenberg ed. (CRC Press, BocaRaton, 1995).

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980. The formation of cross-linked antibodies can target theimmune system to specific types of cells, for example, cancer ordiseased cells expressing OSM-R.beta.

This invention also provides methods of delaying development ofmetastasis in an individual with cancer (including, but not limited to,prostate, lung, breast, ovarian, pancreatic, or colon cancer) using ananti-OSM-R.beta antibody or other embodiments that bind to OSM-R.betalinked to a chemotherapeutic agent. In some embodiments, the antibody isa humanized or chimeric form of a non-human anti-OSM-R.beta antibody.

In yet another aspect, the invention provides methods for preventing ordelaying development of metastases in an individual who has or has had aprimary tumor, comprising administering an effective amount of anOSM-R.beta modulator. This modulator may be given alone or inconjunction with other therapies, such as radiation, chemotherapy, orsurgery. In certain preferred embodiments, the primary tumor has beensubjected to at least one round of surgery, radiation, and/orchemotherapy before administration of the OSM-R.beta modulator. In somecircumstances, the primary tumor may appear to have been completelyremoved by the prior treatment. In other embodiments, the OSM-R.betamodulator is administered prior to or concurrent with treatment of theindividual with surgery, radiation, and/or chemotherapy. In certainparticularly preferred embodiments, that OSM-R.beta modulator is atleast one anti-OSM-R.beta antibody. In other embodiments, these methodsof preventing or delaying growth and/or proliferation of cancer cells invitro or in an individual comprise administering an effective amount ofa composition comprising an anti-OSM-R.beta modulator (such as anantibody that binds to OSM-R.beta) in conjunction with (including linkedto) another therapeutic moiety such as a detectable marker orchemotherapeutic agent to the cell culture or sample, or to theindividual. In some particularly preferred embodiments, the antibody isa humanized or chimeric form of a non-human anti-OSM-R.beta antibody.

In yet another embodiment, the antibody can be employed as adjuvanttherapy at the time of the surgical removal of a cancer expressing theantigen in order to delay the development of metastasis. The antibody orantibody associated with a chemotherapeutic agent can also beadministered before surgery (neoadjuvant therapy) in an individual witha tumor expressing the antigen in order to decrease the size of thetumor and thus enable or simplify surgery, spare tissue during surgery,and/or decrease the resulting disfigurement.

In another embodiment, any of the OSM-R.beta modulators, such as ananti-OSM-R.beta antibody, can be employed as a therapy after thetreatment (e.g., surgical, radiotherapy, chemotherapeutic treatment,etc.) of a primary tumor in order to delay or prevent the development ofmetastases. The OSM-R.beta modulator may be administered alone or linkedto a chemotherapeutic agent. In some embodiments, the antibody is ahumanized or chimeric form of a non-human anti-OSM-R.beta antibody.

In yet another embodiment, any of the OSM-R.beta binding embodimentsdescribed herein can bind to OSM-R.beta-expressing cancerous cells andinduces an active immune response against the cancerous cells expressingOSM-R.beta. In some cases, the active immune response can cause thedeath of the cancerous cells (e.g., antibody binding to cancer cellsinducing apoptotic cell death), or inhibit the growth (e.g., block cellscycle progression) of the cancerous cells. In other cases, any of thenovel antibodies described herein can bind to cancerous cells andantibody dependent cellular cytotoxicity (ADCC) can eliminate cancerouscells to which anti-OSM-R.beta binds. Accordingly, the inventionprovides methods of stimulating an immune response comprisingadministering any of the compositions described herein.

In some cases, antibody binding can also activate both cellular andhumoral immune responses and recruit more natural killer cells orincreased production of cytokines (e.g., IL-2, IFN-g, IL-12, TNF-a,TNF-b, etc.) that further activate an individual's immune system todestroy cancerous cells. In yet another embodiment, the antibody canbind to cancerous cells, and macrophages or other phagocytic cell canopsonize the cancerous cells.

Various formulations of anti-OSM-R.beta antibodies or fragments thereofmay be used for administration. In some embodiments, anti-OSM-R.betaantibodies or fragments thereof may be administered neat. In addition tothe pharmacologically active agent, the compositions of the presentinvention may contain suitable pharmaceutically acceptable carrierscomprising excipients and auxiliaries that are well known in the art andare relatively inert substances that facilitate administration of apharmacologically effective substance or which facilitate processing ofthe active compounds into preparations that can be used pharmaceuticallyfor delivery to the site of action. For example, an excipient can giveform or consistency, or act as a diluent. Suitable excipients includebut are not limited to stabilizing agents, wetting and emulsifyingagents, salts for varying osmolarity, encapsulating agents, buffers, andskin penetration enhancers.

Suitable formulations for parenteral administration include aqueoussolutions of the active compounds in water-soluble form, for example,water-soluble salts. In addition, suspensions of the active compounds asappropriate for oily injection suspensions may be administered. Suitablelipophilic solvents or vehicles include fatty oils, for example, sesameoil, or synthetic fatty acid esters, for example, ethyl oleate ortriglycerides. Aqueous injection suspensions may contain substances thatincrease the viscosity of the suspension and include, for example,sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally,the suspension may also contain stabilizers. Liposomes can also be usedto encapsulate the agent for delivery into the cell.

The pharmaceutical formulation for systemic administration according tothe invention may be formulated for enteral, parenteral or topicaladministration. Indeed, all three types of formulation may be usedsimultaneously to achieve systemic administration of the activeingredient. Excipients as well as formulations for parenteral andnonparenteral drug delivery are set forth in Remington, The Science andPractice of Pharmacy 20th Ed. Mack Publishing (2000).

Suitable formulations for oral administration include hard or softgelatin capsules, pills, tablets, including coated tablets, elixirs,suspensions, syrups or inhalations and controlled release forms thereof.

Generally, these agents are formulated for administration by injection(e.g., intraperitoneally, intravenously, subcutaneously,intramuscularly, etc.), although other forms of administration (e.g.,oral, mucosal, etc) can be also used. Accordingly, anti-OSM-R.betaantibodies are preferably combined with pharmaceutically acceptablevehicles such as saline, Ringer's solution, dextrose solution, and thelike.

The particular dosage regimen, i.e., dose, timing and repetition, willdepend on the particular individual and that individual's medicalhistory. Generally, a dose of at least about 100 ug/kg body weight, morepreferably at least about 250 ug/kg body weight, even more preferably atleast about 750 ug/kg body weight, even more preferably at least about 3mg/kg body weight, even more preferably at least about 5 mg/kg bodyweight, even more preferably at least about 10 mg/kg body weight isadministered.

Empirical considerations, such as the half-life, generally willcontribute to the determination of the dosage. Antibodies, which arecompatible with the human immune system, such as humanized antibodies orfully human antibodies, may be used to prolong half-life of the antibodyand to prevent the antibody being attacked by the host's immune system.Frequency of administration may be determined and adjusted over thecourse of therapy, and is based on reducing the number of cancerouscells, maintaining the reduction of cancerous cells, reducing theproliferation of cancerous cells, or delaying the development ofmetastasis. Alternatively, sustained continuous release formulations ofanti-OSM-R.beta antibodies may be appropriate. Various formulations anddevices for achieving sustained release are known in the art.

In one embodiment, dosages for anti-OSM-R.beta antibodies may bedetermined empirically in individuals who have been given one or moreadministration(s). Individuals are given incremental dosages of ananti-OSM-R.beta antibody. To assess efficacy of anti-OSM-R.betaantibodies, a marker of the specific cancer disease state can befollowed. These include direct measurements of tumor size via palpationor visual observation, indirect measurement of tumor size by x-ray orother imaging techniques; an improvement as assessed by direct tumorbiopsy and microscopic examination of the tumor sample; the measurementof an indirect tumor marker (e.g., PSA for prostate cancer), a decreasein pain or paralysis; improved speech, vision, breathing or otherdisability associated with the tumor; increased appetite; or an increasein quality of life as measured by accepted tests or prolongation ofsurvival. It will be apparent to one of skill in the art that the dosagewill vary depending on the individual, the type of cancer, the stage ofcancer, whether the cancer has begun to metastasize to other location inthe individual, and the past and concurrent treatments being used.

Other formulations include suitable delivery forms known in the artincluding, but not limited to, carriers such as liposomes. See, forexample, Mahato et al. (1997) Pharm. Res. 14:853-859. Liposomalpreparations include, but are not limited to, cytofectins, multilamellarvesicles and unilamellar vesicles.

In some embodiments, more than one antibody may be present. Theantibodies can be monoclonal or polyclonal. Such compositions maycontain at least one, at least two, at least three, at least four, atleast five different antibodies that are reactive against carcinomas,adenocarcinomas, sarcomas, or adenosarcomas. Anti-OSM-R.beta antibodycan be admixed with one or more antibodies reactive against carcinomas,adenocarcinomas, sarcomas, or adenosarcomas in organs including but notlimited to ovary, breast, lung, prostate, colon, kidney, skin, thyroid,bone, upper digestive tract, and pancreas. In one embodiment, a mixtureof different anti-OSM-R.beta antibodies is used. A mixture ofantibodies, as they are often denoted in the art, may be particularlyuseful in treating a broader range of population of individuals.

The following examples are provided to illustrate, but not to limit, theinvention.

EXAMPLES Example 1 Preparation of Cancer Cell Lines as an Immunogen

Whole cells isolated from a human lung adenocarcinoma were used as animmunogen for producing the monoclonal antibodies of this invention.Methods suitable for the practice of this invention are described inU.S. Pat. No. 6,541,225. Generally, to produce monoclonal antibodiesdirected to cell-surface antigens of a specific cell type, it isdesirable to immunize non-transformed B-cells with viable and intactcells of that type, preferably with those cells whose surfaces that arefree of serum. Cells derived from tissue or from cell culture are used.Cell lines that are suitable for the generation of monoclonal antibodiesagainst the antigen OSM-R.Beta, such as but not limited to LUCA38,include: BT-474 (ATCC# HTB-20), MDA-MB-175VII (ATCC# HB-25), MDA-MB-361(ATCC # HB-27), SKBR3 (ATCC# HTB-30), SKMES-1 (ATCC# HTB-58), ES-2(ATCC# CRL-1978), SKOV3 (ATCC# HTB-77), HPAFII (ATCC# CRL-1997), Hs700T(ATCC# HTB-147), Colo205 (ATCC# CCL-222), HT-29 (ATCC# HTB-38), SW480(ATCC# CCL-228), SW948 (ATCC# CCL-237), A498 (ATCC# HTB-44) and Caki-2(ATCC# HTB-47).

The cells were grown in the appropriate nutrient media supplemented withgrowth factors, but free of serum. Immunization with cells that havebeen propagated in a serum-supplemented medium can have extremedisadvantages. Serum contains a complex mixture of small and largebiomolecules with undefined activities. These biomolecules can adhere tothe surfaces of cells and thereby leading to the generation ofantibodies cross-reacting with molecules not representative of thespecific cell type. Additionally, binding of serum biomolecules to thecell surface may lead to the masking of desired cell surface antigentargets. A number of serum-free media preparations are commerciallyknown and publicly available, such as for example, F12/DME (1:1)nutrient media with the following supplements: insulin (10 μg/ml finalconcentration), epidermal growth factor (EGF) (5 ng/ml finalconcentration), selenious acid (2.5×10⁴ M final concentration), andporcine pituitary extract (PPE) (5 μl/ml final concentration).

Under these culture conditions, the cells attached to thesubstrate-coated plates and grew as a monolayer.

To harvest the cells, the cell monolayers were rinsed once with calcium-and magnesium-free Hanks saline solution, incubated in 10 mM EDTA inHanks saline solution at 37C for 15 minutes. The cells were detachedfrom the culture surface by gentle pipetting. The cell suspension waspelleted by centrifugation at 1000×g for 5 minutes. The supernatant wasremoved and cells were resuspended in serum-free medium withnon-denaturing adjuvant as appropriate.

Example 2 Generation of Monoclonal Antibodies

A non-denaturing adjuvant (Ribi, R730, Corixa, Hamilton Mont.) wasrehydrated to 2 ml in phosphate buffered saline. 100 μl of thisrehydrated adjuvant was then gently mixed with some of the cell pelletfrom Example 1 to be used for immunization. Approximately 10⁶ humanfetal kidney cells per mouse were injected into Balb/c mice via footpad,approximately once or twice a week. The precise immunization schedule isas follows: Day zero, immunization plus Ribi. Day 3, immunization plusRibi. Day 7, immunization plus Ribi. Day 24, immunization minus Ribi.Day 29, immunization minus Ribi. Day 32, immunization minus Ribi. Day36, immunization minus Ribi. Day 44, immunization minus Ribi. Day 51,immunization minus Ribi. Day 69, bleed for titer test. Day 71,immunization plus Ribi. Day 74, immunization plus Ribi. Day 81,immunization plus Ribi. Day 91, pre-fusion boost (no Ribi). Day 104,harvest nodes for fusion.

At Day 69, a drop of blood was drawn from the tail of each immunizedanimal to test the titer of antibodies against the cell line used toimmunize using FACS analysis. When the titer reached at least 1:2000,the mice were sacrificed using CO₂ followed by cervical dislocation.Lymph nodes were harvested for hybridoma preparation.

Lymphocytes from mice were fused with the mouse myeloma line X63-Ag8.653using 35% polyethylene glycol 4000. On day 10 following the fusion, thehybridoma supernatants were screened for the presence of the immunizingcells-specific monoclonal antibodies by fluorescence activated cellsorting (FACS). Conditioned medium from each hybridoma was incubated for30 minutes with an aliquot of human fetal kidney cells. Afterincubation, the cell samples were washed, resuspended in 0.1 ml diluentand incubated with 1 μg/ml of FITC conjugated F(ab′)2 fragment of goatanti-mouse IgG for 30 min at 4° C. The cells were washed, resuspended in0.2 ml FACS diluent and analyzed using a FACScan cell analyzer (BectonDickinson; San Jose, Calif.). Hybridoma clones were selected for furtherexpansion, cloning, and characterization based on their binding to thesurface of the human fetal kidney cells by FACS. A hybridoma making amonoclonal antibody designated LUCA38 that binds an antigen designatedAg-LUCA38 was selected.

Example 3 Purification of Anti-OSM-R.beta Antibodies, Including LUCA38

Human lung carcinoma cells were detached from tissue culture flasks inthe presence of 10.0 mM EDTA, centrifuged at 1400 rpm for 5 minutes andresuspended in PBS containing 1% BSA and 2 mM EDTA (FACS diluent). Thecells were counted and adjusted to 10⁷ cells/ml. About 0.1 ml of cellswere incubated with 100-μl hybridoma supernatant in 100 μl FACS diluentfor 30 min at 37° C. Monoclonal antibodies that bind to human fetalkidney cells were purified from tissue culture supernatant usingprotein-G affinity chromatography. The tissue culture supernatant may befirst passed through a bovine IgG column to remove excess bovine IgG inthe supernatant if desired. The following materials were used for theantibody purification process: hybridoma tissue culture supernatant,Immunopure (G) IgG binding buffer (Pierce #21011 Rockford, Ill.),Immunopure IgG Elution Buffer (Pierce #21009), concentrated HCl (foradjusting pH), Corning 1 liter PES (polyether sulfone), 0.22 μm filter(Corning #431098, Corning, N.Y.), Amersham Pharmacia GradiFrac System(Amersham Pharmacia, Piscataway, N.J.), Protein-G Sepharose 4 Fast Flow(AmershamPharmacia #17-0618-02), Stripping buffer which is 3M KSCN/50 mMTris pH 7.8, and PBS (phosphate buffered saline) 3M Tris pH 9.0.

To purify the mouse anti-huOSM-R.beta antibody referred to herein asmu-anti-OSM-R.beta, the volume of supernatant was measured and an equalvolume of binding buffer was added to the supernatant. The mixture wasallowed to equilibrate to room temperature. The supernatant wasclarified by passage through a 0.22 μm filter. The supernatant wasloaded on to a protein-G column using the GradiFrac system (PharmaciaBiotech). The column was washed with 5-10 column volumes of bindingbuffer. The monoclonal antibodies were eluted with the elution bufferand 2 ml fractions were collected. An OD₂₈₀ reading of the fractionswere obtained and the fractions containing monoclonal antibodies werepooled. The eluted monoclonal antibody fractions were neutralized byadding 1/20 volume of 3M Tris. The sample was dialyzed in 1×PBS at 4° C.(with 3 buffer changes of at least 3 hours per change). The purifiedmonoclonal antibodies were sterile filtered (0.2 uM) and stored at 2-8°C.

After purification of the mu-anti-OSM-R.beta monoclonal antibody fromthe hybridoma supernatant, it was re-tested for binding to the humanlung carcinoma cells. The cell samples were prepared as described aboveand incubated with the purified antibody at various concentrations.After incubation the cells were washed, resuspended in 0.1 ml diluentand incubated with 1 μg of FITC conjugated F(ab′)2 fragment of goatanti-mouse IgG for 30 min at 4° C. The cells were washed, resuspended in0.5 ml FACS diluent and analyzed using a FACScan cell sorter (BectonDickinson; San Jose, Calif.). A shift to the right on the FACScanhistogram indicated that the purified antibody still bound to the humanlung carcinoma cells.

In other experiments, the binding of the alpha OSM-R.beta muMAb(referred to herein as mu-anti-OSM-R.beta) to OSM-R.beta was testedusing a live-cell ELISA. The following method was used, although othermethods commonly known in the field are applicable. Cells (SKOV3, SKBR3,SKMES, SW480, HT-29, and HPAF-II—all purchased from the ATCC, Bethesda,Md.) were grown in 10% fetal bovine serum (FBS) containing media toconfluency on tissue culture treated 96-well tissue culture plates(Falcon). Cells were washed free of the culture media and incubated with50 μl of desired antibodies at a desired concentration in Hank'sBalanced Salt Solution (HBSS) containing 1% BSA and 0.1% sodium azidefor 1 hour at room temperature. The cells were then washed three timeswith 100 μl per well of HBSS before an incubation with horseradishperoxidase (HRP) secondary antibody (50 μl per well diluted in HBSS) for30 minutes at room temperature. The cells were finally washed threetimes with HBSS and the color change substrate (TMB substrate, KPL) wasadd to the plate at 100 μl per well. The color change reaction wasstopped with the addition of 100 μl per well of 1M phosphoric acid. Thedeveloped plates were read at O.D. 450.

Example 4 Binding of Mu-Anti OSM-R.beta Antibody to Normal and TumorTissues

Frozen tissue samples from cancer patients were embedded in OCT compoundand quick-frozen in isopentane with dry ice. Cryosections were cut witha Leica 3050 CM mictrotome at thickness of 5 μm and thaw-mounted onvectabound-coated slides. The sections were fixed with ethanol at −20°C. and allowed to air-dry overnight at room temperature. The fixedsections were stored at −80° C. until use. For immunohistochemistry, thetissue sections were retrieved and first incubated in blocking buffer(PBS, 5% normal goat serum, 0.1% Tween 20) for 30 minutes at roomtemperature, and then incubated with the mu-anti-OSM-R.beta and controlmonoclonal antibodies diluted in blocking buffer (1 μg/ml) for 120minutes. The sections were then washed three times with the blockingbuffer. The bound monoclonal antibodies were detected with a goatanti-mouse IgG+IgM (H+L) F(ab′)²-peroxidase conjugates and theperoxidase substrate diaminobenzidine (1 mg/ml, Sigma cat. No. D 5637)in 0.1 M sodium acetate buffer pH 5.05 and 0.003% hydrogen peroxide(Sigma cat. No. H1009). The stained slides were counter-stained withhematoxylin and examined under Nikon microscope.

In some cases, paraffin embedded formaldehyde-fixed tissues were usedfor immunohistochemistry after appropriate antigen retrieval methodswere employed. One such antigen retrieval method is described in Manghamand Isaacson, Histopathology 35:129-33 (1999). Other methods of antigenretrieval and/or detection may be used by one skilled in the art.Results from similar experiments performed using frozen tissues or,where appropriate, fixed tissue with antigen retrieval and polyMICAdetection were performed. The binding of anti-OSM-R.beta antibody to avariety of normal and cancer tissues was assessed. In all cases,antibody binding in control fixed tissues was correlated with that offrozen tissues. The results from frozen tissues were only used if thetwo did not match in the controls.

The results were scored as ‘1+’ for weak positive staining, ‘2+’ formoderate positive staining, ‘3+’ for strong positive staining and ‘−’for negative staining. The results of staining of normal tissues withmu-anti-OSM-R.beta are shown in Table 1. Table 2 shows the binding ofmu-anti-OSM-R.beta antibody to tumor tissue samples.

TABLE 1 Distribution of LUCA38 in normal human tissues Tissue TypeResults Skin +/− basal epithelium (slightly more than negative); +/− to1+ small superficial vessels Kidney 1-2+ rare cells within glomeruliLung +/− to 1+ few alveolar cells (>5%) Pancreas +/− some ducts (30%)Liver Negative Colon Negative Duodenum Negative Breast Negative OvaryNegative Prostate Negative

TABLE 2 Distribution of LUCA38 in human tumor tissues Tissue TypeResults Colon Negative to −/+ Prostate Negative Pancreas +/− to 1+ ontumor Breast +/− to 1+ staining on tumor Ovary +/− to 1+ staining ontumor Kidney +/− to 2+ staining on tumor Lung 1+ to 2+ on tumor

Example 5 Analysis of OSM-R.beta Binding

The binding of the mu-anti-OSM-R.beta antibody LUCA38 to OSM-R.beta wasconfirmed using live cell ELISA. The following method was used, althoughother methods commonly known in the field are applicable. Cells (HT-29,SKOV3, SKMES-1, SW480, SKBR-3, and HPAFII) were grown in 10% fetalbovine serum (FBS) containing media to confluency on tissue culturetreated 96-well plates (Falcon). Cells were washed with PBS and thenincubated with 50 μl of desired antibodies at a desired concentration inHank's Balanced Salt Solution (HBSS) containing 1% BSA and 0.1% sodiumazide for 1 hour at room temperature. The cells were then washed threetimes with 100 μl per well of HBSS before incubation with horseradishperoxidase (HRP) secondary antibody (50 μl per well diluted in HBSS) for30 minutes at room temperature. The cells were finally washed threetimes with HBSS and the color change substrate (TMB substrate, KPL) wasadded to each well at 100 μl per well. The color change reaction wasstopped with the addition of 100 μl per well of 1M phosphoric acid. Theplates were then read at O.D. 450 nm.

In other experiments, mu-LUCA38 was shown to bind a glycoprotein doublet(two fat bands) with the approximate molecular weights of 130-180 kDa.

Example 6 Immunocytochemistry Results

Monoclonal antibody mu-anti-OSM-R.beta (LUCA-38) was used to testreactivity with various cell lines from different types of tissues. Theresults were scored as ‘+’ for weak positive staining, ‘++’ for moderatepositive staining, ‘+++’ for strong positive staining and ‘−’ fornegative staining.

Immunohistochemistry results were obtained using CellArray™ technology,as described in WO 01/43869. Cells from different established cell lineswere removed from the growth surface without using proteases, packed andembedded in OCT compound. The cells were frozen and sectioned, thenstained using a standard IHC protocol.

Results of the binding of the mu-anti-OSM-R.beta antibody LUCA38 tovarious established human normal and tumor cell lines are compiled forconvenience in Table 3. The experiments represented in Table 3 includeLive-cell ELISA (described in Example 4) and CellArray™ bindingexperiments using the methods described herein.

TABLE 3 Immunocytochemistry results Reactivity Reactivity Live Cell Cellline ATCC# Organ Cell Type Cell Array ELISA HMEC CC-2251* Breast Normalmammary epithelial − HuVEC Primary Endothelial cells Normal human adult− BT474 HTB-20 Breast Ductal carcinoma − MCF7 HTB-22 BreastAdenocarcinoma − MDA175 HB-25 Breast Ductal carcinoma − MDA361 HB-27Breast Adenocarcinoma − SK-BR-3 HTB-30 Breast Adenocarcinoma − − 9979RAVEN Lung Lung cancer line + A549 CCL-185 Lung Carcinoma − CA130 RAVENLung Small cell carcinoma − CaLu3 HTB-55 Lung Adenocarcinoma + SKMES1HTB-58 Lung Squamous carcinoma + + ES-2 CRL-1978 Ovary Carcinoma + SKOV3HTB-77 Ovary Adenocarcinoma − + 9926 RAVEN Pancreas Adenocarcinoma −AsPC-1 CRL-1682 Pancreas Adenocarcinoma − HPAFII CRL-1997 PancreasAdenocarcinoma − − Hs700T HTB-147 Pancreas Adenocarcinoma − Colo205CCL-222 Colon Ascites colorectal adenocarcinoma − HT-29 HTB-38 ColonColorectal adenocarcinoma − − SW480 CCL-228 Colon Colorectaladenocarcinoma − + SW948 CCL-237 Colon Colorectal adenocarcinoma − 293CRL-1573 Kidney Transformed with adenovirus DNA − 786-O CRL-1932 KidneyRenal Cell Carcinoma ++ A498 HTB-44 Kidney Carcinoma + Caki2 HTB-47Kidney Clear cell carcinoma − Cos 7 CRL-1651 Kidney (African SV40transformed − Green Monkey) RL65 CRL-10345 Lung (Rat) − SVT2 CCL-163.1Embryo (Mouse) Fibroblast; SV40 transformed − 22RV1 CRL-2505 ProstateCarcinoma − DU145 HTB-81 Prostate Adenocarcinoma − LNCaP CRL-1740Prostate Carcinoma − PC3 CRL-1435 Prostate Adenocarcinoma − TDH-1 RAVENProstate Prostate cancer line − HT1080 Sarcoma + SW872 Sarcoma + Hs746THTB-135 Stomach Carcinoma − NCI-N87 CRL-5822 Stomach Carcinoma −*CC-2251 BioWhittaker

Monoclonal antibody LUCA38 was also used to test reactivity withglioma-derived cell lines. Immunocytochemistry results were obtainedusing similar protocol as described above for the CellArray™ technology.The glioma-derived cell lines were removed from the growth surfacewithout using proteases, packed and embedded in OCT compound. The cellswere frozen and sectioned, then stained using a standard IHC protocol.LUCA38 was positive (+/−to +) on 5/25 glioma-derived cell linesscreened.

Example 7 Effect of Mu-Anti-OSM-R.beta on Renal Cancer Cell Line

The ability of the antibodies to reduce cell number in vitro when grownas a monolayer was assessed using cell monolayers grown in the presenceor absence of varying amounts of test or control purified antibody andthe change in cell number assessed using MTT. MTT is a dye that measuresthe activity of mitochondrial enzymes and correlates with relativeviable cell number. Cells of interest were plated and grown in F12/DMEM(1:1) growth medium supplemented with 10% fetal bovine serum in 96 wellplates. 786-0 cells were plated plated at 500, 1000, 2000 and 4000cells/well in triplicate wells of a 96 well dish: and SKMES-1.Immediately after plating, mu-anti-OSM-R.beta was added. The cells wereincubated at 37° C. in a humidified incubator at 5% CO2/air for 7 days.At the end of the assay, MTT was dissolved in PBS (5 mg/ml) and addeddirectly to wells at 1:10 dilution. Plates were placed back in incubatorfor 4 hours. After the incubation, medium was removed and 100 μl DMSOwas added to solubilize the MTT precipitate. Plates were read at 540 onplate reader.

Mu-anti-OSM-R.beta antibody LUCA38 inhibited the growth of renal cancercell line 786-0 in a dose dependent manner. Results of in vitroexperiments measuring the ability of an anti-OSM-R.beta antibodyaccording to the methods of this example are shown in FIG. 1.

Example 8 Efficacy of Anti-OSM-R.beta Antibody with Human Cells in NudeMice

Human tumor cells were grafted under the kidney capsule in nude (nu/nu)mice. Four tumor cell lines were used. The ES-2 ovarian tumor-derivedcell line, 786-0 renal tumor-derived cell line, and CaLu3-3 lungtumor-derived cell lines were obtained from the ATCC. The 9979 lungadenocarcinoma cell line was derived at Raven biotechnologies, inc. Insome studies both kidneys received xenografts. For the treated animals,grafts were made in the kidney capsule (500 k cells in collagen gel).Anti-OSM-R.beta monoclonal antibody LUCA38 was injectedintraperitoneally at 0.01 mL/g body weight. Dosing was initiated on Day2 following implantation, and doses of LUCA38 or PBS were administeredthree times weekly as single rapid injections. Control mice wereinjected with PBS only. Three days after the final injection, theanimals were euthanized and the kidneys with grafts were examined. Thegrafts and an area around them were then fixed and embedded in paraffinblocks and sectioned through the entire graft area.

The amount of human DNA in the tumors was quantitated using real-timePCR on an Applied Biosystems (Foster City, Calif.) SDS7000 system, withprimers and probe specific for the human ribosomal gene RPL19 accordingto published methods. Each tumor sample was analyzed in triplicate PCRreactions and average DNA concentrations were determined. Average DNAconcentration and standard error of the mean was determined for eachgroup of tumor samples. Statistical significance was determined usingthe Student's T-test (two-tailed, type 1). Tumor growth inhibition wascalculated as the absolute value of [(average tumor volume of treatedgroup/average tumor volume of PBS control group)×100]−100. Table 4 showsxenograft study designs and results.

TABLE 4 LUCA38 SRC Xenograft Model Study Design and Results Cell LineStudied MAb dose (mg/kg) No. animals/group % TGI* 786-0 50 6   6.4 p =0.871 ES-2 50 6 74 p = 0.166 786-0 50 5   58.3 p = 0.107 ES-2 50 5 48 p= 0.224 CaLu3-3 50 4 68 p = 0.173 9979 50 4   30.7 p = 0.040 *TGI =tumor growth inhibition vs. PBS control group; p-values were determinedby the Student's T-test, unpaired.

Example 9 Antitumor Efficacy of LUCA38 in a Subcutaneous Model of HumanOvarian Tumors

This study was designed to test the dose-responsive anti-tumor data forLUCA38 antibody in a subcutaneous model of ovarian cancer.

The ES-2 ovarian tumor-derived cell line was obtained from the ATCC.Cultured cells were trypsinized, washed in media, spun down andresuspended in media at 100 million cells per milliliter of media (5million cells per 0.05 mL volume), then mixed in an equal volume ofMatrigel® for a final injection volume of 0.1 mL. Female CRL. nu/nuhomozygous mice were used. Cells were inoculated by subcutaneousinjection in the back of the neck of the mice. Dosing was initiatedeither on the day of implantation or when tumors were established andmeasurable. For Day 0 dosing, tumors were implanted in the morning andanimals dosed in the afternoon of the same day. For established tumors,animals were randomized among groups as follows: tumor volumes weredetermined, animals were sorted by tumor volume, the mean was determinedand the appropriate number of animals (12 to 15 per group depending onthe model) was selected above and below the mean, removing from thestudy those with small or large tumors. the remaining animals wererandomized by ear tag number into treatment and control groups. Therandom distribution of the final groups was confirmed by T-test (p>0.1was considered randomized).

For each treatment dose group, LUCA38 was diluted in PBS to theappropriate concentration to administer 0.01 mL/g body weight. Doses ofLUCA38 or PBS were administered twice weekly as single rapid injectionsinto the intraperitoneal cavity. Control mice were injected with PBSonly. Dosing was initiated in groups of 15 mice on the day of tumorimplantation.

Tumors were allowed to grow for approximately 6 days prior to initialtumor measurement. Tumors were subsequently measured twice weekly bydigital caliper in three dimensions, and tumor volume was calculated asone-half the product of the three measurements. Tumor volume over timewas the primary endpoint for all studies. Clinical observations weremade daily. Body weight was determined for each animal twice weekly.

Average tumor volumes and standard error of the mean were determined foreach group at each measurement. Statistical significance was determinedusing the Student's T-test (two-tailed, type 1). Tumor growth inhibitionwas calculated as the absolute value of [(average tumor volume oftreated group/average tumor volume of PBS control group)×100]−100. Table5 shows subcutaneous xenograft study designs and results.

TABLE 5 LUCA38 SC Xenograft Model Study Design and Results Cell LineStudied MAb dose (mg/kg) No. animals/group % TGI* ES-2 10 15 49.1% p =0.004 Day 20 30 15 20.5% p = 0.049 Day 20 PBS 15 NA *TGI = tumor growthinhibition vs. PBS control group.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application. Allpublications, patents and patent applications cited herein are herebyincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent or patent applicationwere specifically and individually indicated to be so incorporated byreference.

1. An isolated cell line having ATCC deposit No. PTA-6511.
 2. Anisolated monoclonal antibody produced by hybridoma having ATCC depositNo. PTA-6511.
 3. An isolated antigen-binding fragment of the monoclonalantibody of claim 2, wherein the antigen-binding fragment specificallybinds to oncostatin M receptor beta subunit (OSM-R.beta) expressed onthe surface of a cancer cell.
 4. An isolated antibody that specificallybinds to OSM-R. beta expressed on the surface of a cancer cellcomprising the three complementarity determining regions from the heavychain and the three complementarity determining regions from the lightchain of a monoclonal antibody produced by hybridoma having ATCC depositNo. PTA-6511.
 5. A pharmaceutical composition comprising atherapeutically effective dose of the antibody of claim 4, together witha pharmaceutically acceptable carrier.
 6. The pharmaceutical compositionof claim 5, wherein the composition comprises an additional therapeuticmoiety.
 7. The isolated antibody of claim 4, wherein the isolatedantibody comprises the heavy chain and light chain variable regions froma monoclonal antibody produced by hybridoma having ATCC deposit No.PTA-6511, and the constant regions from a human antibody.
 8. Theisolated antibody of claim 4, wherein the isolated antibody is ahumanized antibody.
 9. The isolated antibody of claim 4, wherein theisolated antibody is an antigen-binding fragment that specifically bindsto OSM-R.beta expressed on the surface of a cancer cell.
 10. An isolatedantibody that specifically binds to OSM-R.beta expressed on the surfaceof a cancer cell, comprising a heavy chain variable region sequence fromthe heavy chain variable region of a monoclonal antibody produced byhybridoma having ATCC deposit No. PTA-6511.
 11. The isolated antibody ofclaim 10, wherein the isolated antibody further comprises a light chainvariable region sequence from the light chain variable region of amonoclonal antibody produced by hybridoma having ATCC deposit No.PTA-6511.
 12. An isolated antibody that specifically binds to OSM-R.betaexpressed on a cancer cell, comprising a light chain variable regionsequence from the light chain variable region of a monoclonal antibodyproduced by hybridoma having ATCC deposit No. PTA-6511.